Seed shattering

ABSTRACT

&#34;The present invention relates to DNA sequences, comprising nucleic acid fragments encoding dehiscence zone-selective proteins, particularly cell wall hydrolases such as polygalacturonases, and their use for modifying dehiscence properties in plants, more particularly pod dehiscence properties in Brassica napus.&#34;

This application is a divisional of Application Ser. No. 09/051,239,filed on Sep. 28, 1998 now U.S Pat. No. 6,420,628, and for whichpriority is claimed under 35 U.S.C. §120. Application Ser. No.09/051,239 is the national phase of PCT International Application No.PCT/EP96/04313 filed on Oct. 4, 1996 under 35 U.S.C. §371. The entirecontents of each of the above-identified applications are herebyincorporated by reference. This application also claims priority ofApplication No. 95402241.4 and 95203328.0 filed in Europe on Oct. 6,1995 and Dec. 8, 1995, respectively under 35 U.S.C. §119.

The present invention relates to DNA sequences, comprising nucleic acidfragments encoding dehiscence zone-selective proteins, particularly cellwall hydrolases such as polygalacturonases, the regulatory regions ofthe corresponding plant genes and their use for modifying dehiscenceproperties in plants, more particularly pod dehiscence properties inBrassica napus.

BACKGROUND OF THE INVENTION

Loss of yield due to seed shedding by mature fruits or pods, also calledpod dehiscence or pod shatter, as well as concomitant increase involunteer growth in the subsequent crop year, are a universal problemwith crops that develop dry dehiscent fruits. An economically importantcrop to which these adverse properties specifically apply is oilseedrape: up to 50% of the potential yield may be lost under adverse weatherconditions.

Dry dehiscent fruits, also commonly called pods, may develop from asingle carpel (such as the legume in many Fabaceae) or from more thanone carpel (such as the silique in many Brassicaceae). In case of thesilique, the pod consists of two carpels joined margin to margin. Thesuture between the margins forms a thick rib, called replum. As podmaturity approaches, the two valves separate progressively from thereplum, eventually resulting in the shattering of the seeds that wereattached to the replum.

Ultrastructural investigation have demonstrated that pod shatter isassociated with the precise degradation of cell wall material at thesite of pod valve separation (i.e., the suture). The degradation of thecell wall and loss of cellular cohesion prior to dehiscence ispredominantly attributed to solubilization of the middle lamella of thecell wall. This middle lamella is found between primary cell walls andis the cement that holds the individual cells together to form a tissue.Cell separation is preceded by an ethylene climacteric, which temporallycorrelates with a tissue-specific increase in the activity of thehydrolytic enzyme cellulase (beta-1,4-glucanase) and this occursspecifically in a layer of cells along the suture, which is called thedehiscence zone. In contrast, the activity of the cell wall degradingenzyme polygalacturonase exhibits no correlation either temporally orspatially with pod dehiscence [Meakin and Roberts (1990), J. Exp. Bot.41; 1003]. Pod dehiscence at an early stage of development ischaracteristic of infestation by the pod midge Dasineura brassicae. Alocalized enhancement of both polygalacturonase and cellulase activityhas been observed. However, regulation of midge-induced andmaturation-associated shatter was found to be different [Meakin andRoberts (1991), Annals of Botany 67: 193].

At first sight, the process of pod dehiscence shares a number offeatures with abscission wherein plants shed organs, such as leaves,flowers and fruits. It has been observed that ethylene induces oraccelerates abscission, whereas auxin inhibits or delays abscission. Adecisive step in abscission is the highly coordinated expression,synthesis and secretion of cell wall hydrolytic enzymes in a discretelayer of cells, called the abscission zone. Cellulases(beta-1,4-glucanases) constitute one class of such cell wall hydrolases.Cellulase activity has been identified in various tissues including leafabscission zones, fruit abscission zones, ripening fruit, senescentcotyledons and styles and anthers [Kemmerer and Tucker (1994), PlantPhysiol. 104: 557 and references therein]. A second class of hydrolasesinvolved in abscission of mainly fruits are polygalacturonases of whichdistinctive isoforms have been identified [Bonghi et al.(1992), PlantMol. Biol. 20: 839; Taylor et al (1990) Planta 183: 133].

Kadkol et al [(1986), Aust. J. Biol. 34: 79] reported increasedresistance towards shattering in a single, Australian accession of rape.Variation in pod maturation has further been observed in mutants of rapestemming from irradiated seeds [Luczkiewicz (1987), Proc. 7th Int.Rapeseed Congress 2: 463]. It can however be concluded that traditionalmethods for breeding have been unsuccessful in introducing shatterresistance into rape cultivars, without interference in other desirabletraits such as early flowering, maturity and blackleg resistance[Prakash and Chopra (1990), Genetical Research 56: 1].

Despite its economic impact very little is known concerning themolecular events and changes in gene expression that occur duringoilseed pod dehiscence. At present, two pod-specific mRNAs whoseexpression is spatially and temporally correlated with pod developmenthave been described. However, the function of the encoded proteins isunknown. [Coupe et al (1993), Plant Mol. Biol. 23: 1223; Coupe et al.(1994), Plant Mol. Biol. 24: 223]. PCT publication WO94/23043 in generalterms describes an approach for regulating plant abscission anddehiscence.

Accordingly, it is an object of the present invention to providedehiscence zone-selective genes in plants.

These and other objects are achieved by the present invention, asevidenced by the summary of the invention, description of the preferredembodiments and claims.

SUMMARY OF THE INVENTION

The present invention provides dehiscence zone(“DZ”)-selective genes ofplants, cDNAs prepared from mRNAs encoded by such genes, and promotersof such genes. In particular this invention provides the cDNA of SEQ IDNO: 1 and the promoter of a gene encoding a mRNA wherein a cDNA of thatmRNA has substantially the nucleotide sequence of SEQ ID NO: 1,particularly the promoter as contained within the 5′ regulatory regionof SEQ ID NO: 14 starting at position 1 and ending at position 2,328.

In another aspect, the present invention also provides DZ-selectivechimeric genes, that can be used for the transformation of a plant toobtain a transgenic plant that has modified dehiscence properties,particularly modified pod-dehiscence properties, when compared to plantsthat do not contain the DZ-selective chimeric gene, due to theexpression of the DZ-selective chimeric gene in the transgenic plant.

In yet another aspect, the present invention thus provides a plantcontaining at least one DZ-selective chimeric gene incorporated in thenuclear genome of its cells, wherein said DZ-selective chimeric genecomprises the following operably linked DNA fragments:

a) a transcribed DNA region encoding:

1) a RNA which, when produced in the cells of a particular DZ of theplant, prevents, inhibits or reduces the expression in such cells of anendogenous gene of the plant, preferably an endogenous DZ-selectivegene, encoding a cell wall hydrolase, particularly anendo-polygalacturonase (an “endo-PG”), or,

2) a protein or polypeptide, which when produced in cells of the DZ,kills or disables them or interferes with their normal metabolism,physiology or development,

b) a plant expressible promoter which directs expression of thetranscribed DNA region at least in cells of the DZ, provided that if thetranscribed DNA region encodes a protein or polypeptide, or encodes anantisense RNA or ribozyme directed to a sense RNA encoded by anendogenous plant gene that is expressed in the plant in cells other thanthose of the DZ, the plant expressible promoter is a DZ-selectivepromoter, i.e., a promoter which directs expression of the transcribedregion selectively in cells of the DZ.

Preferably the transcribed DNA region encodes a protein or polypeptidewhich is toxic to the cells in which it is produced, such as a barnase;a protein or polypeptide that increases the level of auxins or auxinanalogs in the cells in which it is produced, such as a tryptophanmonooxygenase and/or a indole-3-acetamide hydrolase; a protein orpolypeptide that increases the sensitivity to auxin in the cells inwhich it is produced, such as the roIB gene product; or a protein orpolypeptide that decreases the sensitivity to ethylene in the cells inwhich it is produced, such as the mutant ETR1-1 protein or a anotherethylene receptor protein.

In another preferred embodiment of this invention, the transcribed DNAencodes an RNA, such as an antisense RNA or a ribozyme, part of which iscomplementary to the mRNA encoded by a gene which is naturally expressedin the DZ, preferably a DZ-selective gene.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE PRESENTINVENTION

As used herein, the term “dehiscence” refers to the process wherein aplant organ or structure, such as an anther or fruit, opens at maturityalong a certain line or in a definite direction, resulting in theshedding of the content of said organ or structure. In some of itsaspects the process of dehiscence is reminiscent of the process ofabscission, wherein a part or organ, such as a leaf, flower or fruit, isseparated from the rest of the plant.

As used herein, the term “pod” means a dry dehiscent fruit that consistsof one, two or more carpels. In oilseed rape the pod is a bivalvesilique, wherein the valves are delineated by longitudinal dorsal andventral sutures, which comprise the dehiscence zones.

As used herein, the term “pod dehiscence” means the process wherein afruit, particularly a pod, splits open along a discrete layer of cells,eventually resulting in the separation of the valves and subsequentshedding of the seeds contained within the fruit, particularly the pod.Pod dehiscence occurs in a large variety of plants that develop dryfruits, such as in most genera of the Cruciferae.

The term “dehiscence zone” (DZ) in its most general sense includes thetissues in the zone along which a plant organ or structure splits openduring the process of dehiscence. Macroscopically the DZ can usually berecognized by the presence of a clear suture in the organ. In the strictsense the DZ may comprise a region of only 1-3 parenchymatous cellswide. In a pod, this region usually comprises densely packed cells andis adjacent to the periphery of vascular tissue of the replum separatingit from the valve edges. For the purpose of this invention the DZ mayalso include the cell layers surrounding this region. The DZ extendsfrom the locule of the pod to the epidermal suture.

As used herein, the term “promoter” denotes any DNA which is recognizedand bound (directly or indirectly) by a DNA-dependent RNA-polymeraseduring initiation of transcription. A promoter includes thetranscription initiation site, and binding sites for transcriptioninitiation factors and RNA polymerase, and can comprise various othersites (e.g., enhancers), at which gene regulatory proteins may bind.

As used herein, the term “plant-expressible promoter” means a promoterwhich is capable of driving transcription in a plant cell. This includesany promoter of plant origin, but also any promoter of non-plant originwhich is capable of directing transcription in a plant cell, i.e.,certain promoters of viral or bacterial origin such as the CaMV 35S orthe T-DNA promoters.

The term “regulatory region”, as used herein, means any DNA, that isinvolved in driving transcription and controlling (i.e., regulating) thetiming and level of transcription of a given DNA sequence, such as a DNAcoding for a protein or polypeptide. For example, a 5′ regulatory region(or promoter region) is a DNA sequence located upstream (i.e., 5′) of acoding sequence and which comprises the promoter and the 5′-untranslatedleader sequence. A 3′ regulatory region is a DNA sequence locateddownstream (i.e., 3′) of the coding sequence and which comprisessuitable transcription termination (and/or regulation) signals,including one or more polyadenylation signals.

As used herein, the term “cell wall hydrolase” means an enzyme that isinvolved in the degradation of cell wall material, e.g., during theprocess of dehiscence. Examples of such enzymes include, but are notlimited to, polygalacturonase, cellulase (beta-1,4-glucanase),beta-galactosidase, proteases hydrolyzing cell wall proteins, and thelike.

The term “gene” means any DNA fragment comprising a DNA region (the“transcribed DNA region”) that is transcribed into a RNA molecule (e.g.,a mRNA) in a cell under control of suitable regulatory regions, e.g., aplant expressible promoter. A gene may thus comprise several operablylinked DNA fragments such as a promoter, a 5′ untranslated leadersequence, a coding region, and a 3′ untranslated region comprising apolyadenylation site. An endogenous plant gene is a gene which isnaturally found in a plant species. A chimeric gene is any gene which isnot normally found in a plant species or, alternatively, any gene inwhich the promoter is not associated in nature with part or all of thetranscribed DNA region or with at least one other regulatory regions ofthe gene.

The term “expression of a gene” refers to the process wherein a DNAregion under control of regulatory regions, particularly the promoter,is transcribed into an RNA which is biologically active i.e., which iseither capable of interaction with another nucleic acid or which iscapable of being translated into a biologically active polypeptide orprotein. A gene is said to encode an RNA when the end product of theexpression of the gene is biologically active RNA, such as an antisenseRNA or a ribozyme. A gene is said to encode a protein when the endproduct of the expression of the gene is a biologically active proteinor polypeptide.

The phenotypic effect of expression of a gene refers to the biochemical,physiological and/or developmental effects of the production of the RNAor protein, encoded by the gene, on the plant cells (or plants) in whichit is produced. Phenotypic effects of gene expression may be reduced orprevented by reducing or preventing the production of the encoded RNA orprotein, or by otherwise interfering with the biological activity ofsuch RNA or protein.

As defined herein, whenever it is stated in the specification that a“cDNA of such mRNA comprises the nucleotide sequence of SEQ ID NO: X”the RNA thus has the same nucleotide sequence as represented in SEQ IDNO: X except that the U-residues (in the RNA sequence) are replaced byT-residues (in the DNA sequence).

In accordance with this invention, DZ-selective cDNAs and theircorresponding plant genomic DNA fragments are identified as follows:

1) a cDNA library is constructed starting from mRNA isolated from DZtissue and the cDNA library is subjected to differential screening inorder to identify an mRNA which is selectively present in tissues of aparticular DZ when compared to other plant tissues including but notlimited to: pod walls, seeds, replum, leaves, stems, roots, reproductiveorgans, and the like. Alternatively, the cDNA library is screened witholigonucleotides, that are deduced from a determined amino acid sequenceof an isolated protein, such as, for example, a cell wall hydrolase,that is identified to be selectively present in the DZ. Furthermore, itis possible to use the same oligonucleotides in a nested-PCR approachand to use the amplified fragment(s) as a probe to screen the library.The DZ-selective cDNA library can be constructed from a pool of mRNAs,isolated at different stages of DZ development;

2) a cDNA, encoding the DZ-selective mRNA or protein, is isolated andcharacterized;

3) this cDNA is used as a probe to identify and isolate the region inthe plant genome, comprising the nucleotide sequence encoding theDZ-selective mRNA or protein. Alternatively, the genomic DNA can beisolated utilizing inverse PCR using oligonucleotides deduced from thecDNA sequence; and

4) optionally, RNA probes corresponding to the cDNAs are constructed andused in conventional RNA-RNA in-situ hybridization analysis [see e.g.,De Block et al. (1993), Anal. Biochem. 215: 86] of different planttissues, including the particular DZ of interest, to confirm theselective presence of the mRNA produced by the presumed DZ-selectiveendogenous plant gene in that DZ.

The term “dehiscence zone-selective”, with respect to the expression ofa DNA in accordance with this invention, refers to, for practicalpurposes, the highly specific, preferably exclusive, expression of a DNAin cells of one particular DZ, particularly a pod DZ, or a limitedseries of DZs.

Thus a DZ-selective gene is an endogenous gene of a plant that isselectively expressed in the cells of certain dehiscence zones,particularly in the cells of the pod dehiscence zone of the plant. Anyplant which possesses the DZ of interest may be used for the isolationof DZ-selective genes. Suitable plants for the isolation of DZ-selectivegenes are plants of the family Cruciferae including but not limited toArabidopsis thaliana, Brassica campestris, Brassica juncea, andespecially Brassica napus; plants of the family Leguminosae includingbut not limited to Glycine max, Phaseolus vulgaris and the like. ThemRNA (or the cDNA obtained thereof) transcribed from such a gene is aDZ-selective mRNA (or cDNA). A promoter that drives and controls thetranscription of such a mRNA is referred to as a DZ-selective promoter.A DZ-selective promoter can for instance be used to express a cytotoxicgene (e.g., a barnase gene) in a plant so that normal growth anddevelopment, and agronomical performance (as measured for instance byseed yield) of the plant is not negatively affected by expression of thecytotoxic gene in cells other than the DZ cells, preferably in cellsother than the pod DZ cells.

Once the DZ-selective gene (i.e., the genomic DNA fragment, encoding theDZ-selective mRNA from which the DZ-selective cDNA can be prepared) isobtained, the promoter region containing the DZ-selective promoter isdetermined as the region upstream (i.e., located 5′ of from the codoncoding for the first amino acid of the protein encoded by the mRNA. Itis preferred that such promoter region is at least about 400 to 500 bp,preferably at least about 1000 bp, particularly at least about 1500 to2000 bp, upstream of the start codon. For convenience, it is preferredthat such promoter region does not extend more than about 3000 to 5000bp upstream of the start codon. The actual DZ-selective promoter is theregion of the genomic DNA upstream (i.e., 5′) of the region encoding theDZ-selective mRNA. A chimeric gene comprising a DZ-selective promoteroperably linked to the coding region of the gus gene [Jefferson et al.(1986), Proc. Natl. Acad. Sci. USA 83: 8447] will selectively produce,in transgenic plants, detectable beta-glucuronidase activity (encoded bythe qus gene)in the cells of the particular DZ of interest, as assayedby conventional in-situ histochemical techniques [De Block and Debrouwer(1992), The Plant Journal 2: 261; De Block and Debrouwer (1993), Planta189: 218].

Preferred DZ-selective genes from which DZ-selective promoters can beobtained, are genes, preferably Brassica napus genes, that encode aDZ-selective mRNA from which a cDNA can be prepared that contains thesequence corresponding to the sequence of oligonucleotide PG1 (SEQ IDNO: 3) between nucleotide positions 11 and 27 and/or the sequence ofoligonucleotide PG3 (SEQ ID NO: 5) between nucleotide positions 11 and27 (i.e. starting at position 11 and ending at position 27); and/orcontains the sequence complimentary to the oligonucleotide PG2 (SEQ IDNO: 4) between nucleotide positions 11 and 25 and/or the sequence of theoligonucleotide PG5 (SEQ ID NO: 6) between nucleotide positions 11 and27. Preferably, such DZ-selective cDNA contains aforementioned sequencesof oligonucleotides PG1 and PG3 and PG2 and PG5, or encodes a proteinencoded by the region of SEQ ID NO: 1 between nucleotide positions 95and 1,393.

A particularly preferred DZ-selective gene is the Brassica napus genethat encodes a DZ-selective mRNA from which a cDNA can be prepared thatcontains the sequence of SEQ ID NO: 1 at least between nucleotides 10and 1600. Another preferred DZ-selective gene is the Brassica napusgene, that encode a DZ-selective mRNA from which a cDNA can be preparedthat contains the sequence of SEQ ID NO: 11.

A preferred promoter of the present invention is the promoter containedin the 5′ regulatory region of a genomic clone corresponding to the cDNAof SEQ ID NO: 1, e.g. the 5′ regulatory region with the sequence of SEQID NO: 14 starting at position 1 and ending at position 2,328. Aconvenient promoter region is the DNA fragment comprising the sequenceof SEQ ID NO: 14 starting anywhere between the unique SphI site(positions 246-251) and the HindII site (positions 1,836-1,841),particularly between the SphI site and the BamHI site (positions1,051-1,056), and ending at nucleotide position 2,328 (just before theATG translation start codon). Such a promoter region comprises theDZ-selective promoter of the subject invention and the 5′ untranslatedleader region and is used for the construction of DZ-selective chimericgenes. In this regard a particular useful promoter region is the DNAfragment (hereinafter referred to as “PDZ”) with the sequence of SEQ IDNO: 14 between positions 251 (the SphI site) and 2,328.

However, smaller DNA fragments can be used as promoter regions in thisinvention and it is believed that any fragment of the DNA of SEQ ID NO:14 which contains at least the about 490 basepairs, more preferably atleast about 661 basepairs and most preferably about 1326 basepairs,upstream from the translation inititation codon can be used.Particularly preferred smaller fragments to be used as promoter regionin this invention have a DNA sequence comprising the sequence of SEQ IDNO: 14 between nucleotides 1002 and 2328.

It is assumed that DZ-specificity of the promoter of the 5′ regulatoryregion of SEQ ID NO: 14 can be considerably improved by inclusion of thenucleotide sequence of SEQ ID NO: 14 between nucleotides 1002 and 1674.Therefore promoters comprising this nucleotide sequence are particularlypreferred.

Alternatively, artificial promoters can be constructed which containthose internal portions of the promoter of the 5′ regulatory region ofSEQ ID NO: 14 that determine the DZ-selectivity of this promoter. Theseartifical promoters can contain a “core promoter” or “TATA box region”of another promoter capable of expression in plants, such as a CaMV 35S“TATA box region” as described in WO 93/19188. Suitable promoterfragments or artificial promoters can be identified, for example, bytheir approriate fusion to a reporter gene (such as the gus gene) andthe detection of the expression of the reporter gene in the appropriatetissue(s) and at the appropriate developmental stage. It is known thatsuch smaller promoters and/or artificial promoters comprising thoseinternal portions of the 5′ regulatory region of SEQ ID NO:. 14 thatdetermine the DZ selectivity can provide better selectivity oftranscription in DZ-specific cells and/or enhanced levels oftranscription if the transcribed regions of the DZ-selective chimericgenes of the invention.

Besides the actual promoter, the 5′ regulatory region of theDZ-selective gene of this invention also comprises a DNA fragmentencoding a 5′ untranslated leader (5′UTL) sequence of an RNA locatedbetween the transcription start site and the translation start site. Itis believed that the 5′ transcription start site is located betweenposition 2,219 and 2,227 (in SEQ ID NO: 14), resulting in a 5′UTL ofabout 102 to 110 nucleotides in length. It is also believed that thisregion can be replaced by another 5′UTL, such as the 5′UTL of anotherplant-expressible gene, without substantially affecting the specificityof the promoter.

Other useful DZ-selective genes or cDNAs for use in this invention arethose isolated from other sources, e.g., from other cultivars of B.napus or even from other plant species, for instance by using the cDNAof SEQ ID NO: 1 (or SEQ ID NO: 11) as a probe to screen genomiclibraries under high stringency hybridization conditions usingconventional methods as described in Nucleic Acid Hybridization: APractical Approach (1985), IRL Press Ltd UK (Eds. B. D. Hames and S. J.Higgins). A useful gene for the purpose of this invention is thus anygene characterized by encoding a mRNA from which a cDNA variant can beprepared that contains a coding region with a nucleotide sequence thatis essentially similar to that of the coding region of the cDNA clone ofSEQ ID NO: 1, and coding for a protein with polygalacturonase activity.Also promoter regions and promoters can be identified, for example,using such cDNA variants, which are essentially similar to a promoterregion or promoter with a sequence as contained in SEQ ID NO: 14.

With regard to nucleotide sequences (DNA or RNA), such as sequences ofcDNAs or of regulatory regions of a gene, “essentially similar” meansthat when two sequences are aligned, the percent sequence identity—i.e.,the number of positions with identical nucleotides divided by the numberof nucleotides in the shorter of the two sequences—is higher than 80%,preferably higher than 90%, especially with regard to regulatoryregions. The alignment of the two nucleotide sequences is performed bythe Wilbur and Lipmann algorithm [Wilbur and Lipmann (1983), Proc. Nat.Acad. Sci. U.S.A. 80: 726] using a window-size of 20 nucleotides, a wordlength of 4 nucleotides, and a gap penalty of 4.

Two essentially similar cDNA variants will typically encode proteinsthat are essentially similar to each other. For example, a variant ofthe cDNA of SEQ ID NO: 1 will typically encode a protein with an aminoacid sequence which is essentially similar to the amino acid sequence ofthe protein encoded by the cDNA of SEQ ID NO: 1. With regard to “aminoacid sequences”, essentially similar means that when the two relevantsequences are aligned, the percent sequence identity—i.e., the number ofpositions with identical amino acid residues divided by the number ofresidues in the shorter of the two sequences—is higher than 80%,preferably higher than 90%. The alignment of the two amino acidsequences is performed by the Wilbur and Lipmann algorithm [Wilbur andLipmann (1983), Proc. Nat. Acad. Sci. U.S.A. 80: 726] using awindow-size of 20 amino acids, a word length of 2 amino acids, and a gappenalty of 4. Computer-assisted analysis and interpretation of sequencedata, including sequence alignment as described above, can beconveniently performed using the programs of the Intelligenetics™ Suite(Intelligenetics Inc., CA).

In accordance with this invention, the DZ-selective cDNAs and genomicDNAs, as well as the regulatory regions obtained from the genomic DNAsare used to modify the dehiscence properties in plants, particularly poddehiscence properties in Brassica napus.

Thus, in accordance with this invention, a recombinant DNA is providedwhich comprises at least one DZ-selective chimeric gene comprising aplant expressible promoter and a transcribed DNA region, one or both ofwhich is derived from a DZ-selective gene of this invention.

Expression of a DZ-selective chimeric gene in a transgenic plant willhave phenotypic effects only in the cells of the DZ. Thus, expression ofa DZ-selective gene may selectively prevent, suppress, inhibit or reducethe phenotypic effects of expression of endogenous plant genes in acertain dehiscence zone (such as a pod DZ), may selectively kill ordisable cells of the dehiscence zone, or may interfere with the normalmetabolism of DZ cells, resulting in the delay or prevention ofdehiscence, particularly pod dehiscence. For the purpose of thisinvention, a plant cell (such as a DZ cell) is killed or disabled ifeither all biochemical and/or physiological processes of the cell arestopped or, alternatively, if the biochemical and/or physiologicalprocesses of the cell are changed to effectively reduce theextracellular production of at least one enzyme involved in thedegradation of plant cell walls, particularly a pectin degrading enzymesuch as a polygalacturonase, preferably by at least 30%, particularly byat least 75%, more particularly by at least 90%.

For the purpose of the present invention, the phenotypic effects ofexpression of an endogenous gene in a plant cell is prevented,suppressed, inhibited or reduced if the amount of mRNA and/or proteinproduced by the cell by expression of the endogenous gene is reduced,preferably by at least 30%, particularly by at least 75%, moreparticularly by at least 90%.

Plants, in which dehiscence is delayed to different extents, or evenprevented, are produced by transforming a plant with a recombinant DNAcomprising at least one DZ-selective chimeric gene of this inventionwhose expression in the plant results in the production of RNA or aprotein or polypeptide which interferes to different degrees with thenormal functioning of the cells of the dehiscence zone, for example, byreducing the phenotypic effects of expression of one or more endogenousgenes that encode cell wall hydrolytic enzymes, or by killing the DZcells. A delay in the onset of dehiscence, particularly fruitdehiscence, whereby pre-harvest shattering of seeds can be reduced orprevented, will find its application in those plants that suffer frompre-mature (i.e., prior to harvest) seed loss.

In a preferred embodiment of the present invention the DZ-selectivechimeric gene comprises a transcribed DNA region which is transcribedinto an RNA the production of which in the cells of the DZ reduces,inhibits or prevents the expression of an endogenous gene, preferably agene encoding a cell wall hydrolase, particularly anendo-polygalacturonase, in the cells of the DZ. The reduction of theexpression of the endogenous gene can be demonstrated by the reductionof the cytoplasmic levels of the mRNA normally produced by theendogenous gene. The endogenous gene as isolated from the plant willhereinafter be designated as the sense gene which encodes a sense mRNA(or sense pre-mRNA, i.e., an unprocessed mRNA which may include intronregions).

It is preferred that the endogenous sense gene encodes an enzymeinvolved in cell wall hydrolysis, preferably a pectin-degrading enzyme,such as a pectin esterase, a pectin methyl esterase, a pectin lyase, apectate lyase, a polygalacturonase and the like, and particularly anendo-PG. It is believed that pectin degrading enzymes, particularlyendo-polygalacturonases, play an important role in the degradation ofthe middle lamella material of plant cell walls and in the process ofdehiscence, and that selective inhibition of the production of suchenzymes in the dehiscence zone or in the region surrounding thedehiscence zone (e.g., by expression of an antisense RNA to the endo-PGencoding mRNA) on the average delays pod shatter for at least 1 day,preferably 2-5 days.

Although the sense gene may encode any cell wall hydrolase, that issecreted by the cells of the DZ during the process of dehiscence, andthat is involved in the degradation of cell wall material in a certaindehiscence zone, such as for example a cellulase, a glucanase, or abeta-galactosidase, it is preferred that the sense gene is an endogenousDZ-selective gene.

Thus, in one aspect of this invention the DZ-selective chimeric gene ofthis invention encodes an antisense RNA which is complementary to atleast part of a sense mRNA or sense pre-mRNA. Such antisense RNA is saidto be directed to the sense RNA (or sense pre-mRNA). In this regard, theencoded antisense RNA comprises a region which is complementary to apart of the sense mRNA or sense pre-mRNA, preferably to a continuousstretch thereof of at least 50 bases in length, preferably of at leastbetween 100 and 1000 bases in length. The upper limit for the length ofthe region of the antisense RNA which is complementary to the sense RNAis of course the length of the full-length sense pre-mRNA, or to thefull length sense mRNA (which may or may be not processed from a sensepre-mRNA), produced by the plant cells can be used. However, theantisense RNA can be complementary to any part of the sequence of thesense pre-mRNA and/or of the processed sense mRNA: it may becomplementary to the sequence proximal to the 5′ end or capping site, topart or all of the 5′ untranslated region, to an intron or exon region(or to a region bridging an exon and intron) of the sense pre-mRNA, tothe region bridging the noncoding and coding region, to all or part ofthe coding region including the 3′ end of the coding region, and/or toall or part of the 3′ untranslated region. In case the sense gene is amember of a gene family, it is preferred that the antisense RNA encodedby the DZ-selective chimeric gene of this invention contains a sequencewhich is complementary to a region of the sense RNA, e.g., aDZ-selective sense RNA, of at least 50 nucleotides and which has apercent sequence identity (see above) of less than 50%, preferably lessthan 30%, with any region of 50 nucleotides of any sense RNA encoded byany other member of the gene family.

The transcribed DNA region in the DZ-selective chimeric gene of thisinvention can also encode a specific RNA enzyme, or so-called ribozyme(see, e.g., WO89/05852), capable of highly specific cleavage of thesense mRNA or sense pre-mRNA. Such ribozyme is said to be directed tothe sense RNA (or sense pre-mRNA).

Expression of the endogenous gene producing a sense mRNA in a plant canalso be inhibited or repressed by a DZ-selective chimeric gene whichencodes part or all, preferably all, of such sense RNA [Jorgensen et al.(1992), AgBiotech News Info 4: 265N].

The sense RNA to which the antisense RNA or ribozyme encoded by theDZ-selective chimeric gene of this invention is directed is preferably amRNA, wherein a (doublestranded) cDNA of such mRNA comprises thenucleotide sequence of SEQ ID NO: 1 (or SEQ ID NO: 11) or variantsthereof. A preferred region of the sense RNA to which the antisense RNAor ribozyme encoded by the DZ-selective chimeric gene of this inventionis directed comprise a nucleotide sequence of SEQ ID NO:. 1 startinganywhere between nucleotide 890 and 950 and ending anywhere betweennucleotide 1560 and 1620. Another preferred region of the sense RNA towhich the antisense RNA or ribozyme encoded by the DZ-selective chimericgene of this invention is directed comprise a nucleotide sequence of SEQID NO:. 1 starting anywhere between nucleotide 1280 and 1340 and endinganywhere between nucleotide 1560 and 1620, such as, but not limited to,the nucleotide sequence between nucleotides 1296 and 1607.

A DZ-selective chimeric gene encoding a antisense RNA or ribozyme, asdescribed above, is preferably under the control of a DZ-selectivepromoter. Particularly useful DZ-selective promoters are the promotersfrom the DZ-selective genes described above, particularly the promoteras conained within the 5′ regulatory region of SEQ ID NO: 14 betweenposition 1 and 2,328. However, if the DZ-selective gene encodes anantisense RNA and/or ribozyme which is directed to a sense RNA producedby an endogenous DZ-selective gene, preferably a gene encoding aendo-polygalacturonase, it is not required that the promoter of theDZ-selective chimeric gene be a DZ-selective promoter. Nevertheless, insuch case the promoter of the DZ-selective gene should direct expressionat least in cells of the DZ. Indeed, because the sense RNA is producedselectively in the cells of the DZ, the production of the antisense RNAor ribozyme encoded by the DZ-selective gene in cells other than thecells of the DZ, will not have a noticeable phenotypic effect on suchcells. Examples of promoters that direct expression at least in cells ofthe DZ are constitutive plant expressible promoters such as the promoter(P35S) of the 35S transcript of Cauliflower mosaic virus (CaMV)[Guilleyet al. (1982), Cell 30: 763], or the promoter (Pnos) of the nopalinesynthase gene of Agrobacterium tumefaciens [Depicker et al. (1982), J.Mol. Appl Genet. 1: 561].

In another preferred embodiment of this invention, the DZ-selectivechimeric gene encodes an mRNA which, when produced in plant cells, istranslated into a protein or polypeptide which interferes with themetabolism and/or physiology of the plant cells. In most casesproduction of such protein or polypeptide will be undesired in cellsother than the DZ cells and in this regard it is preferred that suchchimeric genes comprise a DZ-selective promoter. Particular usefulDZ-selective promoters are again the promoters from the DZ-selectivegenes described above.

In one aspect of this invention the DZ-selective chimeric gene of thisinvention comprises a transcribed DNA region encoding a protein theactivity of which will result in an increase in biologically activeauxins or auxin analogs within the cells. Such protein may for instancebe involved in auxin biosynthesis, such as tryptophan monooxygenaseand/or the indole-3-acetamide hydrolase, encoded by the Agrobacteriumtumefaciens T-DNA gene 1 (iaaM) and/or gene 2 (iaaH), respectively[Gielen et al. (1984), The EMBO J. 3: 835], or may be theamidohydrolase, encoded by the Arabidopsis thaliana ILR1 gene, whichreleases active indole-3-acetic acid (IAA) from IAA-conjugates [Barteland Fink (1995), Science 268: 1745]. In view of the observed decline inIAA levels prior to pod dehiscence (see Example 1), it is believed thatproduction of such auxin increasing proteins selectively in the DZ cellsof a plant, will not result in the killing of the cells due tooverproduction of IAA, but will rather result in the maintenance and/orrestoration of the IAA levels substantially as found before the observeddecline. This delays the onset of pod dehiscence, through a prolongedinhibition by IAA of production and/or activity of cell wall hydrolyticenzyme normally produced by the cells in the dehiscence zone.

Alternatively the transcribed DNA region of the DZ-selective chimericgene of this invention can comprise the open reading frame of theAgrobacterium rhizogenes roIB gene [Furner et al. (1986), Nature 319:422]. Expression of such DZ-selective chimeric gene in a plant willresult in an increase of the sensitivity of the plant cells towardsauxin through the production of the roIB gene product in cells of thepod DZ thereby countering the normal decline in IAA concentration in theDZ prior to pod shattering.

In another aspect of the present invention, the DZ-selective chimericgene of this invention comprises a transcribed DNA region encoding aprotein, the activity of which results in a decrease of the sensitivitytowards ethylene of the plant cells in which it is produced. Indeed,several genes involved in the ethylene signal transduction pathway inplants have been identified by mutational analysis (e.g. ETR1, ETR2,EIN4, ERS, CTR1, EIN2, EIN3, EIN5, EIN6, HLS1, EIR1, AUX1, EIN7) and fora number of them, the corresponding genes have been cloned [Chang(1996), TIBS 21:129; Bleecker and Schaller (1996), Plant Physiol111:653]. It is thought that ETR1, ETR2, EIN4, ERS all encode ethylenereceptors, while the rest of the genes would be involved in the ethylenesignal transduction pathway downstream of the receptors [Ecker (1995),Science 268: 667]. The ethylene receptors which have been sequenced,bear homology to the receiver domain of the response regulator componentand/or to the histidine protein kinase domain of the sensor component ofthe so-called bacterial two-component regulators and are divided in twoclasses according to the presence or absence of the receiver domainhomology. Class I ethylene receptors comprise both the sensor andreceiver homologous domains and are exemplified by ETR1 (Arabidopsis),and eTAE1 (tomato). Class II ethylene receptors comprise only the domainhomologous to the histidine protein kinase domain of the sensorcomponent and are exemplified by ERS (Arabidopsis) and NR (tomato).Receptors encoded by mutant alleles of the identified genes confer adominant insensitivity to ethylene [Chang (1996), supra; Bleecker andSchaller (1996), supra]. Therefore an example of a DZ-selective chimericgene, comprising a transcribed DNA region encoding a protein whoseactivity results in a decrease of the sensitivity towards ethylene ofthe plant cells in which it is produced, is one which comprises the openreading frame of a dominant, ethylene-insensitive, mutant allele of theArabidopsis thaliana ETR1 gene, such as ETR1-1 [Chang et al. (1993),Science 262: 539]. A plant in which such DZ-selective chimeric gene isexpressed produces a mutant ethylene receptor (the ETR1-1 protein)selectively in the cells of the DZ and these cells therefore becomeinsensitive towards the phytohormone ethylene and do not respond(metabolically) to changes in the concentration of the hormone, such asthe ethylene climacteric observed prior to the onset of pod dehiscence.It is thought that alternatively, a transcribed DNA region comprising anopen reading frame of a dominant, ethylene-insensitive, mutant allele ofany one of the mentioned class I ethylene receptors can be used to thesame effect. In another example of such a DZ-selective chimeric gene,conferring ethylene-insensitivity to the plants cells expressing theDZ-selective chimeric gene, a transcribed DNA region comprising an openreading frame of a dominant, ethylene-insensitive, mutant allele of anyone of the mentioned class II ethylene receptors, such as theArabidopsis thaliana ERS gene [Hua et al. (1995), Science 269: 1712] orthe tomato NR gene [Wilkinson et al. (1995), Science 270:1807] can beused for the same purpose.

It is further assumed that the rest of the products encoded by thegenes, involved in the ethylene signal transduction pathway, mentionedabove, act downstream of the receptors. For CTR1, EIN2 and EIN3 thegenes have been cloned [Ecker (1995), Science 268: 667]. Modulation ofthe expression of the after genes in the dehiscence zone, e.g. byantisense RNA or ribozyme RNA, transcribed under control of aDZ-specific promoter, which is targetted towards the mentioned genes,will also influence sensitivity towards ethylene.

In another aspect of this invention the DZ-selective chimeric gene ofthis invention comprises a transcribed DNA region encoding a protein orpolypeptide which, when produced in a plant cell, such as a cell of apod DZ, kills such cell or at least interferes substantially with itsmetabolism, functioning or development. Examples of such transcribed DNAregions are those comprising DNA sequences encoding ribonucleases suchas RNase T1 and especially barnase [Hartley (1988), J. Mol. Biol.202:913]; cytotoxins such as the A-domain of diphtheria toxin [Greenlandet al. (1983), Proc. Natl. Acad. Sci. USA 80: 6853] or the Pseudomonasexotoxin A. Several other DNA sequences encoding proteins with cytotoxicproperties can be used in accordance with their known biologicalproperties. Examples include, but are not limited to, DNA sequencesencoding proteases such as papain; glucanases; lipases such asphospholipase A2; lipid peroxidases; methylases such as the E. coli Dammethylase; DNases such as the EcoRI endonuclease; plant cell wallinhibitors, and the like.

In still another aspect of this invention the DZ-selective chimeric geneof this invention comprises a transcribed DNA region encoding a proteinor polypeptide which is capable of being secreted from plant cells andof inhibiting at least the activity of at least oneendo-polygalacturonase that is produced in a dehiscence zone (such as apod DZ), particularly the endo-PG encoded by the cDNA of SEQ ID NO: 1.

In the DZ-selective chimeric gene of this invention it is preferred thatthe 5′ untranslated region of encoded RNA is normally associated withthe promoter, such as a DZ-selective promoter, of the chimeric gene.However, the 5′ untranslated region may also be from another plantexpressible gene. Thus, it is preferred that a DZ-selective chimericgene of this invention comprises the complete 5′ regulatory region(including the 5′ untranslated region) of a DZ-selective gene. Aparticularly useful 5′ regulatory region is a region of SEQ ID NO: 14,immediately upstream of position 1,329, preferably a region of at least490 bp, more preferably a region extending to the first SphI siteupstream of position 2,329.

The DZ-selective chimeric genes of this invention preferably alsocomprise 3′ untranslated regions, which direct correct polyadenylationof mRNA and transcription termination in plant cells. These signals canbe obtained from plant genes such as polygalacturonase genes, or theycan be obtained from genes that are foreign to the plants. Examples offoreign 3′ transcription termination and polyadenylation signals arethose of the octopine synthase gene [De Greve et al. (1982), J. Mol.Appl. Genet 1: 499], of the nopaline synthase gene [Depicker et al.(1982), J. Mol. Appl. Genet. 1: 561] or of the T-DNA gene 7 [Velten andSchell (1985), Nucl. Acids Res. 13: 6998] and the like.

Preferably, the recombinant DNA comprising the DZ-selective chimericgene also comprises a conventional chimeric marker gene. The chimericmarker gene can comprise a marker DNA that is; under the control of, andoperatively linked at its 5′ end to, a plant-expressible promoter,preferably a constitutive promoter, such as the CaMV 35S promoter, or alight inducible promoter such as the promoter of the gene encoding thesmall subunit of Rubisco; and operatively linked at its 3′ end tosuitable plant transcription termination and polyadenylation signals.The marker DNA preferably encodes an RNA, protein or polypeptide which,when expressed in the cells of a plant, allows such cells to be readilyseparated from those cells in which the marker DNA is not expressed. Thechoice of the marker DNA is not critical, and any suitable marker DNAcan be selected in a well known manner. For example, a marker DNA canencode a protein that provides a distinguishable color to thetransformed plant cell, such as the A1 gene (Meyer et al. (1987), Nature330: 677), can provide herbicide resistance to the transformed plantcell, such as the bar gene, encoding resistance to phosphinothricin (EP0,242,246), or can provided antibiotic resistance to the transformedcells, such as the aac(6′) gene, encoding resistance to gentamycin(WO94/01560).

The DZ-selective promoters of this invention are believed to be highlyspecific in activity or effect with regard to directing gene expressionin cells of the DZ. However the characteristics (e.g.,tissue-specificity) of a promoter contained in a chimeric gene may beslightly modified in some plants that are transformed with such chimericgene. This can, for example, be attributed to “position effects” as aresult of random integration in the plant genome.

Therefore in some plants transformed with the DZ-selective chimeric geneof this invention low-level expression of the chimeric gene may beobserved in certain non-DZ cells. Thus, optionally, the plant genome canalso be transformed with a second chimeric gene comprising a secondtranscribed DNA region, that is under control of a secondplant-expressible promoter and that encodes a RNA, protein orpolypeptide which is capable of counteracting, preventing or inhibitingthe activity of the gene product of the DZ-selective chimeric gene. Ifthe DZ-selective chimeric gene encodes barnase it is preferred that thesecond chimeric gene encodes a barstar, i.e., an inhibitor of barnase[Hartley (1988), J. Mol. Biol. 202: 913]. Other useful proteins encodedby the second chimeric genes are antibodies or antibody fragments,preferably single chain antibodies, that are capable of specific bindingto the protein encoded by the DZ-selective chimeric gene whereby suchprotein is biologically inactivated.

Preferably the second promoter is capable of driving expression of thesecond transcribed DNA region at least in non-DZ cells of the plant tocounteract, prevent or inhibit the undesired effects of low expressionof the DZ-selective chimeric gene in such cells in some transformedplants. Examples of useful second promoters are the CaMV minimal 35Spromoter [Benfey and Chua (1990), Science 250: 959] or the promoter ofthe nopaline synthase gene of Agrobacterium tumefaciens T-DNA [Depickeret al. (1982), J. Mol. Appl. Genet. 1: 561]. Other useful promoters arepromoters from genes that are known not to be active in the DZ, such asBrassica napus genes encoding a mRNA from which a cDNA can be preparedthat comprises the sequence of SEQ ID. No 8, SEQ ID NO: 10, or SEQ IDNO: 12.

In plants the second chimeric gene is preferably in the same geneticlocus as the DZ-selective chimeric gene so as to ensure their jointsegregation. This can be obtained by combining both chimeric genes on asingle transforming DNA, such as a vector or as part of the same T-DNA.However, in some cases a joint segregation is not always desirable.Therefore both constructs can be present on separate transforming DNAs,so that transformation might result in the integration of the twoconstructs at different location in the plant genome.

In still a further embodiment of the present invention, a plant withmodified dehiscence properties can be obtained from a single plant cellby transforming the cell in a known manner, resulting in the stableincorporation of a DZ-selective chimeric gene of the invention into thenuclear genome.

A recombinant DNA comprising a DZ-selective chimeric gene can be stablyincorporated in the nuclear genome of a cell of a plant, particularly aplant that is susceptible to Agrobacterium-mediated transformation. Genetransfer can be carried out with a vector that is a disarmed Ti-plasmid,comprising a DZ-selective chimeric gene of the invention, and carried byAgrobacterium. This transformation can be carried out using theprocedures described, for example, in EP 0,116,718. Ti-plasmid vectorsystems comprise a DZ-selective chimeric gene between the T-DNA bordersequences, or at least to the left of the right T-DNA border.Alternatively, any other type of vector can be used to transform theplant cell, applying methods such as direct gene transfer (as described,for example, in EP 0,233,247), pollen-mediated transformation (asdescribed, for example, in EP 0,270,356, WO85/01856 and U.S. Pat. No.4,684,611), plant RNA virus-mediated transformation (as described, forexample, in EP 0,067,553 and U.S. Pat. No. 4,407,956), liposome-mediatedtransformation (as described, for example, in U.S. Pat. No. 4,536,475),and the like.

Other methods, such as microprojectile bombardment as described, forexample, by Fromm et al. [(1990), Bio/Technology 8: 833] and Gordon-Kammet al. [(1990), The Plant Cell 2: 603], are suitable as well. Cells ofmonocotyledonous plants, such as the major cereals, can also betransformed using wounded or enzyme-degraded intact tissue capable offorming compact embryogenic callus, or the embryogenic callus obtainedthereof, as described in WO92/09696. The resulting transformed plantcell can then be used to regenerate a transformed plant in aconventional manner.

The obtained transformed plant can be used in a conventional breedingscheme to produce more transformed plants with the same characteristicsor to introduce the DZ-selective chimeric gene of the invention in othervarieties of the same or related plant species. Seeds obtained from thetransformed plants contain the DZ-selective chimeric gene of theinvention as a stable genomic insert.

The following Examples describe the isolation and characterization of aDZ-selective gene from Brassica napus, the identification ofDZ-selective promoter, and the use of such a promoter for themodification of dehiscence properties in plants. Unless stated otherwisein the Examples, all recombinant DNA techniques are carried outaccording to standard protocols as described in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al.(1994) Current Protocols in Molecular Biology, Current Protocols, USA.Standard materials and methods for plant molecular work are described inPlant Molecular Biology Labfax (1993) by R. D. D. Croy, jointlypublished by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications, UK.

In the examples and in the description of the invention, reference ismade to following sequences of the Sequence Listing:

SEQ ID NO: 1 DZ-selective cDNA encoding a endo-polygalacturonase ofBrassica napus SEQ ID NO: 3 oligonucleotide PG1 SEQ ID NO: 4oligonucleotide PG2 SEQ ID NO: 5 oligonucleotide PG3 SEQ ID NO: 6oligonucleotide PG5 SEQ ID NO: 7 PCR Fragment BPG32-26 SEQ ID NO: 8 PCRFragment KPG32-8 SEQ ID NO: 9 PCR Fragment LPG12-16 SEQ ID NO: 10 PCRFragment LPG32-24 SEQ ID NO: 11 PCR Fragment LPG32-25 SEQ ID NO: 12 PCRFragment LPG32-32 SEQ ID NO: 13 T-DNA of pGSV5 SEQ ID NO: 14 sequence ofgenomic clone comprising the DZ-selec- tive promoter region drivingexpression of an endopolygalacturonase gene of Brassica napus

In order to further illustrate the present invention and advantagesthereof, the following specific examples are given, it being understoodthat the same are intended as illustrative and in nowise limitative.

EXAMPLE 1

Characterization of Pod Dehiscence During Pod Development.

Endogenous Phytohormone Profiles During Pod Development.

Brassica napus cv Fido plants were grown in an unheated greenhouse. At12 days after germination plants were transferred to, and further grownin, 1000 cm³ compost. Pods were collected at one week intervals from twoto eight weeks after anthesis. The pods were taken from the base of theterminal one of the first three axillary racemes. The pods wereseparated into dehiscence zone, pod wall and seeds.

The samples were grinded with a mortar and pestle and then extracted for16 hours at −20 C. in a total volume of 80% methanol. Purification andanalysis of phytohormones was carried out essentially as described[Bialek and Cohen (1989), Plant Physiol. 90: 398; Prinsen et al. (1991),in: A Laboratory Guide for Cellular and Molecular Plant Biology. Ed.Negrutiu and Gharti-Chhetri. Birkhäuser Verlag, Basel/Boston/Berlinpp.175-185, pp.323-324; Chauvaux et al. (1993), J. Chromatogr. A 657:337].

Different parts of the pods (pod wall, dehiscence zone and seeds) werescreened for endogenous concentrations of the ethylene precursor1-aminocyclopropane-1-carboxylic acid (ACC) and conjugates thereof aswell as for indole-3-acetic acid (IAA) and conjugates thereof.

A peak in ethylene evolution [see also Meakin and Roberts (1990), J.Exp. Bot. 41: 1003] was observed immediately before pod shattering; thispeak was correlated with observed peaks of free ACC. Especially in thedehiscence zone a decline in IAA concentrations (free as well asconjugated forms) was observed, just before the onset of pod opening.This decline in IAA concentration was specifically correlated with anincreased cellulase activity in the dehiscence zone.

In a further experiment ethylene production was inhibited by treatingthe pods with aminoethoxyvinylglycine (AVG), a competitive inhibitor ofthe enzyme ACC-synthase. AVG was applied 28 days after anthesis at 500mg/l. This treatment resulted in a 40-50% reduction of ethyleneproduction in the entire pod and was accompanied by a delay of pod wallsenescence of approximately 4 days. Decreased endogenous ACCconcentrations in both dehiscence zone and seeds of the treated podscorrelated with the reduced ethylene production. In the other tissuesanalysed (pod wall, septum and the zone between dehiscence zone and podwall) no such decrease in ACC concentrations or synthesis could bedemonstrated. A decrease in endogenous IAA content in the dehiscencezone preceding pod opening was also observed in these experiments bothin control and in AVG-treated plants.

To examine the auxin involvement in pod shattering, the synthetic auxin4-chlorophenoxyacetic acid (4CPA) was used to manipulate auxin levels.4CPA was applied 35 days after anthesis as a spray at 150 mg/l in orderto artificially keep auxin concentration at a high level during theentire period. This resulted in a distinct retardation in pod shattertendency (see Table 1), as well as a delay of pod wall senescence ofabout two weeks. No effect was observed on the endogenous phytohormoneconcentrations. Beta-Glucanase activity however was markedly decreasedin the dehiscence zone. These results are clearly indicative of theinhibitory effect of auxins on the production and/or activity ofbeta-glucanase.

The decline in auxin is a major trigger of pod shatter.

TABLE 1 Force (in 10⁻³N) needed to initiate and propagate pod opening asmeasured in the Cantilever bending test [Kadkol et al. [(1986), Aust. J.Bot. 34: 595] with pods (8% moisture) of oilseed rape cv Fido. Testedplants were either untreated, sprayed with AVG to reduce ethylenevalues, or sprayed with 4CPA to prevent the auxin drop. (SED: StandardError on Difference; df: degree of freedom) SED untreated AVG 4CPA (27df) To initiate 143.6 170.8 194.9 14.4  crack To propagate 167.1 171.4222.6 17.21 crack

Demonstration of Polygalacturonate-degrading Enzyme Activity in PodDehiscence Zones

Pods of oilseed rape cv Fido were harvested at 6.5 weeks after anthesis,stripped of the carpels and seeds, and crude enzyme extracts wereprepared from tissues surrounding the dehiscence zones, including thereplum with a vascular bundle and the thin membrane separating the twolocules of the silique. Extracts were subsequently tested with respectto their action against polymeric substrates (uronic acids), usingmolecular weight down-shift assays, based on gel-permeationchromatography of substrate incubated with a boiled (used as reference)and active enzyme preparation respectively. The assay in particulardetects enzymes with endo-activity as removal of single monosaccharidesin an exo-fashion only changes molecular weight distribution of thepolymeric substrate very slowly. Analysis for uronic acids was carriedout essentially as described by Blumenkrantz and Asboe-Hansen [(1973),Anal. Biochem. 54: 484]. The assay was used here only to demonstrate thepresence of enzyme activities in a strictly qualitative sense.

DZ preparations from oilseed rape pods contain all enzyme activitiesrequired for a full depolymerization of pectic polymers of low degree ofmethylation. It was found that one component of the enzyme mixture wasspecifically acting on polygalacturonate polymers. It was furtherdemonstrated that only endo-polygalacturonase among known plant enzymesis responsible for the molecular weight down-shift of thepolygalacturonate preparations used.

It can be concluded that endo-polygalacturonase plays an important rolein the extensive degradation of middle lamella material observed duringpod dehiscence.

Anatomical Observations During the Process of Dehiscence

Detailed examination of the structure of pod tissues has given moreinsight in the anatomical changes associated with the biochemicalprocesses that take place in the dehiscence zone. It was observed byelectron microscopy that rapid dehydration of the pod wall immediatelyprecedes the degradation of parenchymatous cells situated in thedehiscence zone, mesocarp, septum and in the seed abcission zone.Initial signs of breakdown were shown by swelling of the cell walls.Subsequent cell-separation was seen only in the dehiscence zone, and wasobserved to take place along the line of the middle lamella to befollowed by the dispersion of the microfibrils of the cell wall.Finally, all the cells of the dehiscence zone were observed to separatewhile the two valves of the pod remained attached only by the vascularstrands which pass through the dehiscence zone. Analysis using electronmicroscopy revealed very dramatic degradation of the middle lamelladuring pod opening while the primary cell wall was left essentiallyintact but for some thinning and softening processes. These observationsindicate that any processes in the primary cell wall are accessory tothe degradation of the middle lamella.

The complete dissolution of the middle lamella of cells in thedehiscence zone indicates the presence of pectin degrading enzymes suchas endoPG. While such enzymes degrade charged portions of the middlelamella pectins, other polysaccharide hydrolases, with affinity towardsneutral polymers, are involved to complete the depolymerization of themiddle lamella.

A beta-galactanase and a beta-glucanase were purified to homogeneity.Detailed investigation of the substrate specificity indicated that theseenzymes are involved in thinning of the primary cell wall in thedeiscence zone.

EXAMPLE 2

Isolation of a DZ-selective Endo-Polygalacturonase cDNA Clone fromBrassica Napus.

Poly-A⁺ mRNA of pod dehiscence zones of Brassica napus cv Topaz plantswas prepared as follows. Twenty grams of tissue (leaves, dehiscencezones, pod walls, roots or stems) were ground in liquid nitrogen andhomogenized for 30 seconds in a Waring blender with 100 ml of extractionbuffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5%sarkosyl, 0.1 M 2-mercaptoethanol). The homogenate was transferred to afresh tube and 1/10 volume of 2 M sodium acetate, pH 4.0, and 1 volumeof TE saturated phenol/chloroform was added. The solution was shakedvigorously, cooled on ice for 15 min. and centrifuged at 10,000×g for 15min at 4 C. The supernatant was re-extracted with phenol/chloroform asdescribed above. An equal volume of isopropanol was added to there-extracted supernatant and RNA was precipitated by an overnightincubation at −20 C. After centrifugation at 10,000×g for 15 min, theRNA pellet was dissolved in 2 ml of denaturation buffer. Fourteen ml of4 M LiCl was then added and the solution kept in an ice-bath overnight.The RNA was pelleted by centrifugation at 10,000×g for 15 min, washed in80% ethanol, dried and dissolved in 1 ml of water. Poly-A⁺ RNA wasisolated on an oligo-d(T) sepharose column according to themanufacturer's guidelines (Boehringer, Mannheim).

Random or oligo-d(T) primed first strand cDNA synthesis was performedusing M-MLV reverse transcriptase and 6 μg of total poly-A⁺ RNA asprepared above according to conditions outlined by the manufacturer(Life Technologies/BRL). First strand cDNAs were used as template DNAfor further PCR reactions.

Four degenerated primers were designed based on conserved regions frompublished polygalacturonase (PG) amino acid sequences from tomato[DellaPenna et al. (1986), Proc. Natl. Acad. Sci. USA 83:6420; Griersonet al. (1986), Nucl. Acids Res. 14: 8595], maize [Niogret et al (1991),Plant Mol. Biol. 17: 1155], avocado and Oenothera [Brown and Crouch(1990), The Plant Cell 2: 263]. The sequences of the four primers used(PG1, PG2, PG3 and PG5) are shown in SEQ ID NOS: 3-6. A restrictionenzyme site for EcoRI was introduced at the 5-end of the two upstreamprimers PG1 and PG3 and a BamHI site was introduced at the 5-end of thetwo downstream primers PG2 and PG5.

All PCR reactions had the following final composition: 50 mM KCl, 10 mMTris-HCl, pH 8.3, 1.5 mM MgCl, and 0.001% (w/v) gelatin, 100 pmoles ofdegenerated primers and 1U of Taq DNA polymerase in a 50 μl reactionvolume. After an initial denaturation of template DNA at 95° C. for 3minutes in 1×PCR reaction buffer, the PCR reaction was initiated byadding 1U of Taq DNA polymerase in 1×PCR buffer (hot start PCR) usingthe following conditions: 1 min. at 95° C., 1 min. at 45° C. and 1 min.at 72° C. for 35 cycles followed by 72° C. for 3 min. For hot startnested PCR 2 μl of a PCR reaction was applied as template in a new PCRreaction. The PCR products were chloroform extracted and ethanolprecipitated, redissolved in TE and digested with the restrictionenzymes BamHI and EcoRI. The restricted PCR products were purified fromlow melting agarose, and cloned into pGEM-7z cut with BamHI and EcoRI.DNA sequences of the PCR fragments were obtained by the dideoxy chaintermination method using Sequenase version 2.0 (Pharmacia).

The longest PCR fragment was obtained by using the PG1/PG5 primercombination. Hot start nested PCR was performed with the PG3/PG2,PG1/PG2 or PG3/PG5 primer combinations using a small aliquot of thePG1/PG5 PCR reaction as a template. Seven highly divergent PG-relatedclones were identified by sequencing of the PCR products, indicating thepresence of at least seven different PG isoforms. Three forms wereobtained from a single tissue only, namely Ipg32-25 (SEQ ID NO: 11) fromdehiscence zones, kpg32-8 (SEQ ID NO: 8) from pod walls and bpg32-26(SEQ ID NO: 7) from leaves. Lpg32-32 (SEQ ID NO: 12), Ipg32-24 (SEQ IDNO: 10) were found only in the two pod tissues, whereas Ipg12-16 (SEQ IDNO: 9) was obtained from all three tissues analyzed. It should be noted,that Ipg35-8 (containing the DNA sequence of SEQ ID NO: 1 from position884 to 1,245) was the only type identified in the dehiscence zone whenthe PG3/PG5 primer combination was used in a nested PCR reaction.

The expression of the PG-related PCR clone Ipg35-8 in roots, stems,leaves and hypocotyls as well as during pod development was investigatedby Northern analysis as follows. Total RNA of individual tissues wasseparated by gel electrophoresis in 0.66 M formaldehyde/1% agarose gel[Sambrook et al. (1989), supra]. RNA was transferred onto Hybond-Nfilters and fixed to the filter by UV-irradiation. The filters wereprehybridized for4 hours in 5×Denhardt, 25 mM Na₂HPO₄, 25 mM NaH₂PO₄,0.1% pyrophosphate, 750 mM NaCl, 5 mM EDTA and 100 μg/ml denaturedherring sperm DNA at 68° C. The PCR products were radioactively labelledand were heat-denatured and added directly to the pre-hybridizationbuffer and hybridization was then continued for 16 hours at 68° C. Thefilter was washed according to Sambrook et al. [(1989), supra] where thefinal wash was carried out at 68° C. in 0.2×SSC, 0.1% SDS. The filterswere autoradiographed at −80° C. using an intensifying screen.

No transcripts hybridizing to the Ipg35-8 clone could be detected intotal RNA isolated from roots, stems, leaves and hypocotyls However, theIpg35-8 clone hybridized to a 1.6-1.7 kb transcript that is exclusivelyexpressed in the dehiscence zone during all stages analyzed and wasfound to increase dramatically in abundance after week 5.

A DZ-selective cDNA library was constructed in Lambda ZAP® II insertionvectors (Stratagene) using 5 μg of poly-A⁺ RNA isolated from dehiscencezones 6 weeks after anthesis. cDNAs larger than 1 kbp were purified froma low temperature melting agarose gel and ligated into the Lambda ZAP®II vector. The primary library consisted of 1.25×10⁶ pfu with anaveraged cDNA insert size of app. 1.5 kbp. Library screening was doneaccording to standard procedures at high stringency [Sambrook et al(1989), supra].

cDNAs were sequenced using Sequenase v. 2.0 (Amersham). Sequenceanalysis was performed with the GCG sequence analysis software packagev. 7 [Devereux et al. (1984), Nucl. Acids Res. 12: 387].

Screening 300,000 plaques with the Ipg35-8 PCR-fragment as probe gaveapproximately 200 positive hybridization signals. Five stronglyhybridizing plaques were purified to homogeneity. After excision of theinsert DNA from the lambda vector, restriction enzyme analysis showedthe cDNA inserts to be approximately 1600 bp in all cDNA clones exceptone, which only had an insert of 1300 bp. Restriction enzyme mapping ofthe 4 largest cDNA inserts (designated as X, 5, 9 and 11 respectively)showed minor differences between the 4 cDNA inserts.

Most noteworthy is the presence of a Nsil restriction enzyme site incDNA clones X and 11 and the presence of a HindII site in cDNA clone 9.In contrast, none of these restriction sites are present in cDNA clone5. Partial sequencing of the 5 and 3 cDNA ends revealed additionalsequence variations including small deletions/insertions between thedifferent cDNA clones. These results indicate the expression in thedehiscence zone of different but highly homologous PG-encoding genes.The sequence data also showed that the larger 4 cDNA inserts allcontained the complete coding sequence for the PG protein.

The complete sequence of cDNA clone X and the deduced amino acidsequence of its largest open reading frame is shown in SEQ ID NO: 1. Theopen reading frame encodes a protein of 433 amino acids in size with anestimated molecular weight of 46.6 kD and with considerable similarityto known endo-polygalacturonases. Similar to other cell wall hydrolasesthe presumed DZ-selective endo-PG is initially produced as a precursorcontaining a N-terminal signal peptide which is cleaved offco-translationally. The most likely cleavage site is located betweenamino acids 23 and 24 and gives rise to a mature protein with anestimated molecular weight of 44.2 kD.

Northern analysis, using cDNA clone X as a probe, confirmed and extendedthe previously obtained expression pattern. Total RNA was prepared asdescribed from different tissues of the pods (the dehiscence zone, thepod walls, seeds and septum) at 5 time points (2, 3, 5, 7 and 9 weeksafter anthesis—WAA). 5 μg of total RNA was seperated bygel-electrophoresis and hybridized with the radiolabelled cDNA of SEQ IDNO: 1 as a probe under the stringent conditions descibed above. Theautoradiogram was developed after overnight exposure. At 2 WAA, nosignal was detectable; at 3 WAA a faint signal was observed; based ondensitometry scannings and readings, the expression level measured attime point 5 WAA is about 3.5× the amount seen at 3 WAA, at 7 WAA isabout 7× the amount seen at 3 WAA, at 9 WAA is about 12 times the amountseen at 3 WAA. No signal was detected in the pod walls or seeds. Faintexpression (comparable with the level in the DZ at 3 WAA) was measuredin the septum at 9 WAA.

The RNA used in this experiment has been extracted from the respectivetissues of plants for which the pod development took about 9 weeks.

EXAMPLE 3

Isolation of a DZ-selective Promoter from a B. napus Genomic CloneCorresponding to the cDNA Clone Ipq 35-8

A commercially available lambda EMBL3 Brassica napus cv. Bridger genomiclibrary (Clontech Laboratories, Inc.) in Escherichia coli strain NM538was screened as follows. After transfer to Hybond-N nylon membranes, theIpg35-8 cDNA was radioactively labelled using random priming, and thefilters were hybridized under high stringency conditions in 5×SSPE,5×Denhardt, 0,5% SDS, 50 μg/ml herring DNA (1×SSPE: 0,18 M NaCl, 10 mMsodium phosphate, pH 7,7, 1 mM EDTA) and washed under high stringencyconditions (68° C., 0,1×SSPE, 0,1% SDS in the final wash). Approximately600,000 plaques were screened and eleven hybridizing plaques wereisolated. Two hybridizing plaques, lambda 2 and 11, were rescreenedtwice. Following the second rescreening phage lysates were made fromlambda 2 and 11 on E. coli NM538 grown without maltose. DNA preparationsfrom lambda 2 and lambda 11 were digested with SalI, subjected to gelelectrophoresis and transferred to Hybond-N nylon membrane.Hybridization with the labelled Ipg35-8 cDNA clone, resulted inidentical hybridization patterns for both clones. A strongly hybridizing6.3 kb SalI fragment was isolated from lambda 11 and inserted intopUC18, resulting in the master clone 6.3SaI. In order to confirm 6.3SaIas corresponding to Ipg35-8, a sequencing primer was designed enablingthe determination of a DNA stretch encoding two unique amino acidspresent in Ipg35-8. Dideoxy sequencing by the Sanger method confirmedthat the isolated genomic clone 6.3SaI was in this respect identical tothe Ipg35-8 cDNA. Restriction mapping of this clone demonstrated that itcovered the entire Ipg35-8 open reading frame and contained moreoverapp. 100 to 200 bp of downstream sequence and app. 3.5 kb of upstreamsequence. The DNA sequence of a stretch of about 2.3 kb (including thepromoter, the 5′ untranslated region and the first 24 nucleotides of theopen reading frame) was determined and is presented in SEQ ID NO: 14.

In view of the fact that the cDNA clone (SEQ ID NO: 1) and the genomicclone (SEQ ID NO: 14) were isolated from different B. napus cultivars(resp. cv.Topaz and cv.Bridger), it was surprisingly found that uponalignment of both sequences the overlapping fragment displayed 100%sequence identity.

The transcription start site of the DZ selective gene corresponding tothe gene contained in the 6.3SaI clone is determined using generallyknown techniques such as primer extension analysis [Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, NY] or RACE-PCR [Innis et al. (1990) PCRProtocols: A Guide to Methods and Applications, Academic Press Inc.].The 5′UTL is believed to be located between positions 2,219 and 2,227 ofSEQ ID NO: 14.

Using well-established site-directed mutagenesis techniques [Ausubel etal. (1994), supra], the DNA sequence is modified to create a uniquerestriction enzyme (e.g., NcoI) recognition site around the ATGtranslation initiation codon of the coding sequence. This allows astraightforward fusion of the promoter region of the DZ-selective geneto a DNA sequence of interest to construct a DZ-selective chimeric geneof the invention. Using a unique restriction enzyme recognition sitelocated between 500 to 2000 base pairs upstream (i.e., 5′) of the uniquerestriction site surrounding the ATG translation initiation codon, awell defined DNA fragment is isolated, that is subsequently used as apromoter cassette, hereinafter referred to as PDZ, that directsDZ-selective expression in plants.

For example, a SphI-NcoI fragment (of about 2.08 kb), which is capableof directing DZ-selective expression in plants, is then subsequentlyused as a promoter cassette, hereinafter referred to as PDZ1.

A DZ-selective chimeric gene (PDZ or PDZ1-gus-3′nos) is constructedcomprising the following operably linked DNA fragments:

PDZ or PDZ1: the 5′ regulatory region comprising a DZ-selectivepromoter,

gus: a DNA fragment coding for beta-glucuronidase [Jefferson et al.(1986) Proc. Natl. Acad. Sci. USA 83: 8447];

3′nos: the 3′ untranslated end comprising the polyadenylation site ofthe nopaline synthase gene (“3′nos”)[Depicker et al. (1982), J. Mol.Appl. Genet. 1: 561].

A second promoter cassette which is capable of directing DZ-selectiveexpression in plants, was obtained using well-established site-directedmutagenesis techniques to modify the DNA sequence to create a uniquerestriction site immediately upstream of the ATG translation initiationcodon of the coding sequence. For this purpose a SmaI site has beenengineered, by changing the A-nucleotides of SEQ ID NO:. 14 at positions2327 and 2328 into G-nucleotides . The SphI-SmaI fragment of about 2.1kb, hereinafter referred to as promoter cassette PDZ2, was fused at theSmaI site upstream of the GUS coding region in the plasmid pBI101(Clontech Laboratories, Inc CA, USA), resulting in a plasmid(2.1guspgem7) carrying the chimeric PDZ2-gus-3′nos gene construct.

A chimeric selectable marker gene PSSU-bar-3′ocs was constructed [DeAlmeida et al. (1989), Mol. Gen. Genet. 218: 78]. It comprises thefollowing operably linked DNA fragments:

PSSU: the promoter region of Arabidopsis thalianaribulose-1,5-biphosphate carboxylase small subunit 1A encoding gene[Krebbers et al. (1988), Plant Mol. Biol. 11: 745),

bar: the region of the bar gene encoding phosphinothricin acetyltransferase[Thompson et al. (1987), The EMBO J. 6: 2519],

3′ocs: a 3′ untranslated end comprising the polyadenylation site of theoctopine synthase gene [De Greve et al. (1983), J. Mol. Appl. Genet. 1:499].

Alternatively, a PSSU-bar-3′g7 was constructed comprising identicalfragments as the preceding chimeric selectable marker gene, except thatthe 3′ocs was replaced by the 3′ untranslated end comprising thepolyadenylation site of the T-DNA gene 7 (3′g7; Velten and Schell(1985), Nucl. Acids Research, 13, 6981).

Both the DZ-selective chimeric gene (PDZ2-gus-3′nos; cloned as aHindIII-XhoI fragment of about 4.2 kb) and the chimeric marker gene(PSSU-bar-3′g7) were introduced into the polylinker located between theborder sequences of the T-DNA vector pGSV5, resulting in plasmid vectorpTCO155 carrying the PDZ2-gus-3′nos and pSsuAra-bar-3′g7 chimeric geneconstructs between the T-DNA border repeats. pGSV5 was derived fromplasmid pGSC1700 [Cornelissen and Vandewiele (1989), Nucl. Acids Res.17: 833] but differs from the latter in that it does not contain abeta-lactamase gene and that its T-DNA is characterized by the sequenceof SEQ ID NO: 13.

EXAMPLE 4

Construction of a Chimeric-Gene Carrying the Barnase Coding Region UnderControl of the Endo-PG Promoter

A DZ-selective chimeric gene (PDZ-barnase-3′nos) is constructedcomprising the following operably linked DNA fragments:

PDZ: the 5′ regulatory region of Example 3, comprising a DZ-selectivepromoter,

barnase: a DNA fragment coding for barnase of Bacillus amyloliquefaciens[Hartley (1988), J. Mol. Biol. 202: 913],

3′nos

Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs chimericmarker gene are introduced into the polylinker located between theborder sequences of the T-DNA vector pGSV5 of Example 4.

PDZ2-barnase-3′nos between T-DNA border repeats was constructed byreplacing the TA29 promoter upstream of the barnase coding region inpTC099, by the PDZ2 promoter cassette. To this end the 2.1 kb (blunted)SphI-SmaI fragment comprising PDZ2 was fused with its SmaI site to theblunted NcoI site overlapping with the ATG-codon which had beenengineered at the 5′end of the coding sequence for the mature barnase inpTCO99, resulting in the plasmid vector pTPR1 carrying thePDZ2-barnase-3′nos chimeric gene between the T-DNA border repeats. TheT-DNA vector part of pTCO99 is derived from that of pGSV5 by insertionof an EcoRI linker (GGAATTCC) into the SmaI site of the polylinker, anda BgIII linker (CAGATCTG) into the NcoI site of the polylinker followedby introduction of the chimeric pTA29-barnase-3′nos gene of pTCO113[WO96/26283] into the EcoRI site of the polylinker. Introduction of thechimeric selectable marker gene pSSUAra-bar-3′g7 in the polylinkersequence of pTPR1 between the T-DNA border repeats results in pTPR3.

An additional T-DNA vector (pTPR2) is constructed wherein theDZ-selective chimeric gene described above (PDZ2-barnase-3′nos) isaccompanied by the BglII fragment of pTCO113 [WO96/26283] comprising thebarstar coding region under control of the nopaline synthase promoter(pnos-barstar-3′g7) inserted into the polylinker of pTPR1 between theT-DNA border repeats. Introduction of the chimeric selectable markergene pSSUAra-bar-3′g7 in the polylinker sequence of pTPR2 between theT-DNA border repeats results in pTPR4.

EXAMPLE 5

Construction of a DZ-Selective Chimeric Gene Encoding T-DNA Gene 1Product or the roIB Gene Product

A DZ-selective chimeric gene (PDZ-g1-3′nos) is constructed comprisingthe following operably linked DNA fragments:

PDZ or PDZ1 or PDZ2: the 5′ regulatory region of Example 3, comprising aDZ-selective promoter,

g1: a DNA fragment encoding the Agrobacterium tumefaciens tryptophan2-monooxygenase (iaaM or T-DNA gene 1 product)[Gielen et al. (1984),EMBO J. 3: 835], obtained by polymerase chain reaction usingappropriately desigend primers comprising sequences respectivelyidentical and complementary to the sequences immediately flanking gene1.

3′nos

A second DZ-selective chimeric gene (PDZ-g2-3′nos) is constructedcomprising the following operably linked DNA fragments:

PDZ or PDZ1 or PDZ2: the 5′ regulatory region of Example 3, comprising aDZ-selective promoter,

g2: a DNA fragment encoding the Agrobacterium tumefaciensindole-3-acetamide hydrolase (iaaH or T-DNA gene 2 product)[Gielen etal. (1984), EMBO J. 3: 835], obtained by polymerase chain reactionamplification, using appropriately desigend primers comprising sequencesrespectively identical and complementary to the sequences immediatelyflanking gene 2.

3′nos

Both the DZ-selective chimeric gene (either PDZ-g1-3′nos alone or incombination with PDZ-g2-3′nos) and the PSSU-bar-3′ocs or PSSU-bar-3′g7chimeric marker gene are introduced into the polylinker located betweenthe border sequences of the T-DNA vector pGSV5 of Example 4.

Another DZ-selective chimeric gene (PDZ-roIB-3′nos) is constructedcomprising the following operably linked DNA fragments:

PDZ: the 5′ regulatory region of Example 3, comprising a DZ-selectivepromoter,

roIB: the open reading frame of the Agrobacterium rhizogenes roIB gene[Furner et al. (1986), Nature 319: 422]

3′nos

Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs or thePSSU-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

EXAMPLE 6

Construction of a DZ-Selective Chimeric Gene Encoding a Mutant ETR1-1Ethylene Receptor

A DZ-selective chimeric gene (PDZ-etr1-1-3′nos) is constructedcomprising the following operably linked DNA fragments:

PDZ or PDZ1 or PDZ2: the 5′ regulatory region of Example 3, comprising aDZ-selective promoter,

etr1-1: the open reading frame of the dominant, ethylene-insensitivemutant allele of the Arabidopsis thaliana ETR gene [Chang et al. (1993),Science 262: 539], isolated as 2.7 kb fragment comprising the exons ofthe coding sequence seperated by 5 introns obtained by PCR amplificationusing the plasmid carrying the 7.3 kb genomic EcoRI fragment comprisingthe DNA of the mutant etr1 allele [Chang et al. (1993), Science 262:539] and appropriately designed primers.

3′nos

Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs orPSSUAra-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

EXAMPLE 7

Construction of a DZ-Selective Chimeric Gene Encoding Antisense RNAComplementary to mRNA from Which the cDNA of SEQ ID NO: 1 can BePrepared

A DZ-selective chimeric gene (PDZ-anti-PG-1-3′nos) is constructedcomprising the following operably linked DNA fragments:

PDZ or PDZ1 or PDZ2: the 5′ regulatory region of Example 3, comprising aDZ-selective promoter,

anti-PG-1: a DNA fragment encoding an RNA which is complementary to theRNA encoded by the region of SEQ ID NO: 1 between nucleotide positions10 and 1600.

3′nos

To this end the CaMV35S promoter of the 35S-antisense PG constructcomprising a DNA sequence complementary to the complete sequence of theSEQ ID NO: 1 cloned between a CaMV 35S promoter and a polyadenylationsignal (as described below) was eliminated by digestion with HincII andXhoI, and replaced by the fragment comprising PDZ2.

Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs orPSSU-bar-3′ocs chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

Another DZ-selective chimeric gene (PDZ-anti-PG-2-3′nos) is constructedcomprising the following operably linked DNA fragments:

PDZ or PDZ1 or PDZ2: a 5′ regulatory region of Example 3, comprising aDZ-selective promoter,

anti-PG-2: a DNA fragment encoding an RNA which is complementary to theRNA encoded by the region of SEQ ID NO: 1 between nucleotide positions20 and 700.

3′nos

Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs or thePSSU-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

Still another DZ-selective chimeric gene (PDZ-anti-PG-3-3′nos) isconstructed comprising the following operably linked DNA fragments:

PDZ or PDZ1 or PDZ2: the 5′ regulatory region of Example 3, comprising aDZ-selective promoter,

anti-PG-3: a DNA fragment encoding an RNA which is complementary to theRNA encoded by the region of SEQ ID NO: 1 between nucleotide positions800 and 1600.

3′nos

Both the DZ-selective chimeric gene and the PSSU-bar-3′ocs or thePSSUAra-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

Three other antisense constructs were constructed comprising a CaMV 35Spromoter, a DNA sequence complementary to the complete sequence of SEQID NO: 1, or a DNA sequence complementary to 679 bp of the 3′end of SEQID NO: 1 (A67), or a DNA sequence complementary to 336 bp of the 3′endof SEQ ID NO: 1 (A30), and a polyadenylation signal. The cDNA ofcDNA-library clone X was excised as a EcoRI-XhoI fragment and insertedin Bluescript® (Stratagene, Calif. USA). The full length cDNA wasisolated as a BamHI-XhoI fragment from this plasmid and inserted intoBamHI-XhoI digested pRT100 vector [Topfer et al (1987) Nucleic AcidResearch 15, 5890], between the CaMV35S promoter and polyadenylationsignal. The resulting plasmid was digested with BamHI and EcoRI, treatedwith Klenow polymerase and self-ligated.

The HaeIII-XhoI fragment of the Bluescript® plasmid with the cDNA insertcomprising the 3′ end 679 bp of SEQ ID NO: 1 was inserted into theSmaI-XhoI digested pRT100 vector, between the CaMV35S promoter andpolyadenylation signal, resulting in plasmid A67.

The A67 construct was digested with Xbal and StyI, treated with Klenowpolymerase and self-ligated, resulting in plasmid A30, comprising theDNA sequence complementary to 336 bp of the 3′ end of SEQ ID NO: 1between the CaMV35S promoter and polyadenylation signal. The chimericgenes were isolated as PstI fragments.

35S-antisense-PG chimeric genes and the PSSU-bar-3′ocs or thePSSU-bar-3′g7 chimeric marker gene are introduced into the polylinkerlocated between the border sequences of the T-DNA vector pGSV5 ofExample 4.

EXAMPLE 8

Transformation of Oilseed Rape and Characterization of theTransformants.

Agrobacterium-Mediated Transformation.

Hypocotyl explants of Brassica napus are obtained, cultured andtransformed essentially as described by De Block et al. [(1989), PlantPhysiol. 91: 694), except for the following modifications:

hypocotyl explants are precultured for 3 days in A2 medium [MS, 0.5 g/lMes (pH5.7), 1.2% glucose, 0.5% agarose, 1 mg/l 2,4-D, 0.25 mg/lnaphthalene acetic acid (NAA)and 1 mg/l 6-benzylaminopurine (BAP)].

infection medium A3 is MS, 0.5 g/l Mes (pH5.7), 1.2% glucose, 0.1 mg/lNAA, 0.75 mg/l BAP and 0.01 mg/l gibberellinic acid (GA3).

selection medium A5 is MS, 0.5 g/l Mes (pH5.7), 1.2% glucose, 40 mg/ladenine.SO₄, 0.5 g/l polyvinylpyrrolidone (PVP), 0.5% agarose, 0.1mg/NAA, 0.75 mg/l BAP, 0.01 mg/l GA3, 250 mg/l carbenicillin, 250 mg/ltriacillin, 0.5 mg/l AgNO₃.

regeneration medium A6 is MS, 0.5 g/l Mes (pH5.7), 2% sucrose, 40 mg/ladenine.SO₄, 0.5 g/l PVP, 0.5% agarose, 0.0025 mg/l BAP and 250 mg/ltriacillin.

healthy shoots are transferred to rooting medium which was A8: 100-130ml half concentrated MS, 1% sucrose (pH5.0), 1 mg/l isobutyric acid(IBA), 100 mg/l triacillin added to 300 ml perlite (final pH6.2) in 1liter vessels.

MS stands for Murashige and Skoog medium [Murashige and Skoog (1962),Physiol. Plant. 15: 473).

Hypocotyl explants are infected with Agrobacterium tumefaciens strainC58C1Rif^(R) carrying:

a helper Ti-plasmid pMP90 [Koncz and Schell (1986), Mol. Gen. Genet 204:383) or a derivative thereof (such as pGV4000), which is obtained byinsertion of a bacterial chloramphenicol resistance gene linked to a 2.5kb fragment having homology with the T-DNA vector pGSV5, into pMP90.

T-DNA vector pGSV5 containing between the T-DNA borders the DZ-selectivechimeric gene of Example 3, 4, 5, 6, or 7 and the chimeric marker gene.

Selected lines from these transformants harboring one type of thechimeric genes of the invention are further used for crossingexperiments, yielding new lines comprising combinations of the chimericgenes of the invention.

Characterization of Transformants

Transformed Brassica napus plants of Example 8, comprising in theirnuclear genomes the DZ-selective chimeric gene of Example 3, arecharacterized with respect to the presence of beta-glucuronidase (GUS)activity in various tissues of the plants using conventional in-situhistochemical techniques [De Block and Debrouwer (1992), The PlantJournal 2:261; De Block and Debrouwer (1993), Planta 189: 218]. GUSactivity is only found in the tissues of the pod DZ, attesting to thefact that the DZ-selective promoter of Example 3 directs expressionselectively in the pod DZ.

Transformed Brassica napus plants of Example 8, comprising in theirnuclear genomes the DZ-selective chimeric genes of either Example 4, 5,6 or 7 alone or in combination are characterized with respect to thefollowing characteristics:

1) changes in physiological processes by analysing diminution ofexpression of targetted gene products (such as cell wall hydrolases) ordiminution in the biochemical activities (see Blumenkratz, supra), bymonitoring the heterologous gene expression (see, Sambrook et al supra),or by measuring the endogenous levels of IAA and IAA conjugates duringdevelopment (see, Example 1)

2) changes in DZ anatomy and DZ cell walls during pod senescence bylight microscopy and transmission electron microscopy; the extent ofcell seperation after pod opening by analysing seperated DZ surfaceswith the scanning electron microscope (See, Example 1)

3) changes in the mechanical properties of the DZ and their seed shatterresistance by analysing the shatter resistance of individual pods. Thiscan be done by the cantilever test as described by Kadkol et al.[(1986), Aust. J. Bot. 34: 595]. Clamped pods are loaded as a cantileverin a “universal testing machine”, consisting of a cross-head beam movedby actuators to which a load cell applies a constant force to deflectthe pod. This records the displacement and the force necessary toinitiate and propagate an opening in the pod dehiscence zone.Alternatively, a first assessment of shatter susceptibility is carriedout with detached pods subjected to controlled vibration (simulatingimpact with canopy and machinery). The vibration consists of horizontaloscillation of fixed amplitute in a container with steel balls toenhance energy transfer. In yet another procedure, susceptibility tocrack propagation is determined by friction measurement. In this case,the force generated due to friction between a wedge forced along the DZis recorded and enables comparison of DZ tissues in selected, extremeexamples of resistance.

Finally, individual selected lines are subjected to per se performanceanalysis in the field. The design of these field trials is based on thecultivation of individual lines (homozygous for the transgene) at twolocations in three replicates.

Analysis of a statistically significant number of pods from differenttransformed plants demonstrates an increase in pod shatter resistancewhen compared to untransformed control plants.

Needless to say, the use of the DZ-selective promoter and recombinantDNA constructs of this invention is not limited to the transformation ofthe specific plant of the examples. Such promoter and recombinant DNAconstructs can be useful in transforming any crop, where the promotercan drive gene expression, preferably where such expression is to occurabundantly in the plant cells of the dehiscence zone.

Also, the use of the DZ-selective promoter of the present invention isnot limited to the control of particular transcribed DNA regions of theinvention, but can be used to control expression of any foreign gene orDNA fragment in a plant.

Furthermore, the present invention is not limited to the specificDZ-selective promoter described in the above Examples. Rather, thepresent invention encompasses promoters, equivalent to the one of theExamples, which can be used to control the expression of a structuralgene, at least substantially selectively in the plant cells of thedehiscence zone. Indeed, the DNA sequence of the DZ-selective promoterof the Examples can be modified by replacing some of its nucleotideswith other nucleotides and/or deleting or inserting some nucleotides,provided that such modifications do not alter substantially the timing,level and tissue-specificity of expression controlled by the promoter,as measured by GUS assays in transgenic plants transformed with achimeric qus gene under control of the modified promoter (see, Example3). Up to 20% of the nucleotides of a promoter may be changed withoutaffecting the characteristics of the promoter. Such promoters can beisolated by hybridization under standard conditions (Sambrook et al,supra) using selected DNA fragments of SEQ ID NO: 14, as describedabove.

All publications (including patent publications) cited in thisapplication are hereby incorporated by reference.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 14 <210> SEQ ID NO 1 <211> LENGTH: 1631<212> TYPE: DNA <213> ORGANISM: Brassica napus <220> FEATURE:<223> OTHER INFORMATION: Location 95-163 = regi#on encoding the presumed       endo-PG signal peptide.<223> OTHER INFORMATION: Location 884-900 = reg #ion of the endo-PG cDNA      corresponding to oligonucleotide PG3<223> OTHER INFORMATION: Location 1059-1073 = r#egion of the endo-PG cDNA       complementary to oligonucleotide PG2<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (95)..(1393)<223> OTHER INFORMATION: Location 1229-1245 = r#egion of the endo-PG cDNA       complementary to oligonucleotide PG5<223> OTHER INFORMATION: Location 821-837 = reg #ion of endo-PG cDNA      corresponding to oligonucleotide PG1. <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (1439)<223> OTHER INFORMATION: Strain cv. Topaz.<223> OTHER INFORMATION: n = a, c, g, t,  #any, other, unknown, or other<400> SEQUENCE: 1ggcacgagaa aaactgcaaa gagtctcata ttagttctta ctctcaagaa tc#aaacacac     60 tctttctaaa aagattagcg tttcaaaccc cgaa atg gcc cgt t#gt ttt gga agt    115                    #                  #  Met Ala Arg Cys Phe Gly Ser                    #                  #    1               # 5 cta gct gtt ttc tta tgc gtt ctt ttg atg ct#c gct tgc tgc caa gct      163Leu Ala Val Phe Leu Cys Val Leu Leu Met Le #u Ala Cys Cys Gln Ala         10          #         15          #         20ttg agt agc aac gta gat gat gga tat ggt ca#t gaa gat gga agc ttc      211Leu Ser Ser Asn Val Asp Asp Gly Tyr Gly Hi #s Glu Asp Gly Ser Phe     25              #     30              #     35gaa tcc gat agt tta atc aag ctc aac aac ga#c gac gac gtt ctt acc      259Glu Ser Asp Ser Leu Ile Lys Leu Asn Asn As #p Asp Asp Val Leu Thr 40                  # 45                  # 50                  # 55ttg aaa agc tct gat aga ccc act acc gaa tc#a tca act gtt agt gtt      307Leu Lys Ser Ser Asp Arg Pro Thr Thr Glu Se #r Ser Thr Val Ser Val                 60  #                 65  #                 70tcg aac ttc gga gcc aaa gga gat gga aaa ac#c gat gat act cag gct      355Ser Asn Phe Gly Ala Lys Gly Asp Gly Lys Th #r Asp Asp Thr Gln Ala             75      #             80      #             85ttc aag aaa gca tgg aag aag gca tgt tca ac#a aat gga gtt act act      403Phe Lys Lys Ala Trp Lys Lys Ala Cys Ser Th #r Asn Gly Val Thr Thr         90          #         95          #        100ttc tta att cct aaa gga aag act tat ctc ct#t aag tct att aga ttc      451Phe Leu Ile Pro Lys Gly Lys Thr Tyr Leu Le #u Lys Ser Ile Arg Phe    105               #   110               #   115aga ggc cca tgc aaa tct tta cgt agc ttc ca#g atc cta ggc act tta      499Arg Gly Pro Cys Lys Ser Leu Arg Ser Phe Gl #n Ile Leu Gly Thr Leu120                 1 #25                 1 #30                 1 #35tca gct tct aca aaa cga tcg gat tac agt aa#t gac aag aac cac tgg      547Ser Ala Ser Thr Lys Arg Ser Asp Tyr Ser As #n Asp Lys Asn His Trp                140   #               145   #               150ctt att ttg gaa gac gtt aat aat cta tca at#c gat ggc ggc tcg gcg      595Leu Ile Leu Glu Asp Val Asn Asn Leu Ser Il #e Asp Gly Gly Ser Ala            155       #           160       #           165ggg att gtt gat ggc aac gga aat atc tgg tg#g caa aac tca tgc aaa      643Gly Ile Val Asp Gly Asn Gly Asn Ile Trp Tr #p Gln Asn Ser Cys Lys        170           #       175           #       180atc gac aaa tct aag cca tgc aca aaa gcg cc#a acg gct ctt act ctc      691Ile Asp Lys Ser Lys Pro Cys Thr Lys Ala Pr #o Thr Ala Leu Thr Leu    185               #   190               #   195tac aac cta aag aat ttg aat gtg aag aat ct#g aga gtg aga aat gca      739Tyr Asn Leu Lys Asn Leu Asn Val Lys Asn Le #u Arg Val Arg Asn Ala200                 2 #05                 2 #10                 2 #15cag cag att cag att tcg att gag aaa tgc aa#c aat gtt ggc gtt aag      787Gln Gln Ile Gln Ile Ser Ile Glu Lys Cys As #n Asn Val Gly Val Lys                220   #               225   #               230aat gtt aag atc act gct cct ggc gat agt cc#c aac acg gat ggt att      835Asn Val Lys Ile Thr Ala Pro Gly Asp Ser Pr #o Asn Thr Asp Gly Ile            235       #           240       #           245cat atc gtt gct act aaa aac att cga atc tc#c aat tca gac att ggg      883His Ile Val Ala Thr Lys Asn Ile Arg Ile Se #r Asn Ser Asp Ile Gly        250           #       255           #       260aca ggt gat gat tgt ata tcc att gag gat gg#a tcg caa aat gtt caa      931Thr Gly Asp Asp Cys Ile Ser Ile Glu Asp Gl #y Ser Gln Asn Val Gln    265               #   270               #   275atc aat gat tta act tgc ggc ccc ggt cat gg#g atc agc att gga agc      979Ile Asn Asp Leu Thr Cys Gly Pro Gly His Gl #y Ile Ser Ile Gly Ser280                 2 #85                 2 #90                 2 #95ttg ggg gat gac aat tcc aaa gct tat gta tc#g gga att gat gtg gat     1027Leu Gly Asp Asp Asn Ser Lys Ala Tyr Val Se #r Gly Ile Asp Val Asp                300   #               305   #               310ggt gct acg ctc tct gag act gac aat gga gt#a aga atc aag act tac     1075Gly Ala Thr Leu Ser Glu Thr Asp Asn Gly Va #l Arg Ile Lys Thr Tyr            315       #           320       #           325cag gga ggg tca gga act gct aag aac att aa#a ttc caa aac att cgt     1123Gln Gly Gly Ser Gly Thr Ala Lys Asn Ile Ly #s Phe Gln Asn Ile Arg        330           #       335           #       340atg gat aat gtc aag aat ccg atc ata atc ga#c cag aac tac tgc gac     1171Met Asp Asn Val Lys Asn Pro Ile Ile Ile As #p Gln Asn Tyr Cys Asp    345               #   350               #   355aag gac aaa tgc gaa cag caa gaa tct gcg gt#t caa gtg aac aat gtc     1219Lys Asp Lys Cys Glu Gln Gln Glu Ser Ala Va #l Gln Val Asn Asn Val360                 3 #65                 3 #70                 3 #75gtg tat cag aac ata aaa ggt acg agc gca ac#a gat gtg gcg ata atg     1267Val Tyr Gln Asn Ile Lys Gly Thr Ser Ala Th #r Asp Val Ala Ile Met                380   #               385   #               390ttt aat tgc agt gtg aaa tat cca tgc caa gg#t att gtg ctt gag aat     1315Phe Asn Cys Ser Val Lys Tyr Pro Cys Gln Gl #y Ile Val Leu Glu Asn            395       #           400       #           405gtg aac atc aaa gga gga aaa gct tct tgc ga#a aat gtc aat gtt aag     1363Val Asn Ile Lys Gly Gly Lys Ala Ser Cys Gl #u Asn Val Asn Val Lys        410           #       415           #       420gat aaa ggc act gtt tct cct aaa tgc cct ta#attactaa gctgattatg       1413 Asp Lys Gly Thr Val Ser Pro Lys Cys Pro    425               #   430taatatacat aaatacgtag tatatntaat tatagatgca tgtatatcgt ta#tctacgta   1473ttgattcttg atatatatag aaaactaaag atatatggga atatacatac aa#tagttgag   1533ataattgttg tcttgtatat gattcactga agttgattgc ttgtccatga at#aaatgaat   1593 aatatcattt ctctaaaaaa aaaaaaaaaa aaaaaaaa      #                   #   1631 <210> SEQ ID NO 2 <211> LENGTH: 433<212> TYPE: PRT <213> ORGANISM: Brassica napus <220> FEATURE:<223> OTHER INFORMATION: Strain cv. Topaz. <400> SEQUENCE: 2Met Ala Arg Cys Phe Gly Ser Leu Ala Val Ph #e Leu Cys Val Leu Leu  1               5  #                 10  #                 15Met Leu Ala Cys Cys Gln Ala Leu Ser Ser As #n Val Asp Asp Gly Tyr             20      #             25      #             30Gly His Glu Asp Gly Ser Phe Glu Ser Asp Se #r Leu Ile Lys Leu Asn         35          #         40          #         45Asn Asp Asp Asp Val Leu Thr Leu Lys Ser Se #r Asp Arg Pro Thr Thr     50              #     55              #     60Glu Ser Ser Thr Val Ser Val Ser Asn Phe Gl #y Ala Lys Gly Asp Gly 65                  # 70                  # 75                  # 80Lys Thr Asp Asp Thr Gln Ala Phe Lys Lys Al #a Trp Lys Lys Ala Cys                 85  #                 90  #                 95Ser Thr Asn Gly Val Thr Thr Phe Leu Ile Pr #o Lys Gly Lys Thr Tyr            100       #           105       #           110Leu Leu Lys Ser Ile Arg Phe Arg Gly Pro Cy #s Lys Ser Leu Arg Ser        115           #       120           #       125Phe Gln Ile Leu Gly Thr Leu Ser Ala Ser Th #r Lys Arg Ser Asp Tyr    130               #   135               #   140Ser Asn Asp Lys Asn His Trp Leu Ile Leu Gl #u Asp Val Asn Asn Leu145                 1 #50                 1 #55                 1 #60Ser Ile Asp Gly Gly Ser Ala Gly Ile Val As #p Gly Asn Gly Asn Ile                165   #               170   #               175Trp Trp Gln Asn Ser Cys Lys Ile Asp Lys Se #r Lys Pro Cys Thr Lys            180       #           185       #           190Ala Pro Thr Ala Leu Thr Leu Tyr Asn Leu Ly #s Asn Leu Asn Val Lys        195           #       200           #       205Asn Leu Arg Val Arg Asn Ala Gln Gln Ile Gl #n Ile Ser Ile Glu Lys    210               #   215               #   220Cys Asn Asn Val Gly Val Lys Asn Val Lys Il #e Thr Ala Pro Gly Asp225                 2 #30                 2 #35                 2 #40Ser Pro Asn Thr Asp Gly Ile His Ile Val Al #a Thr Lys Asn Ile Arg                245   #               250   #               255Ile Ser Asn Ser Asp Ile Gly Thr Gly Asp As #p Cys Ile Ser Ile Glu            260       #           265       #           270Asp Gly Ser Gln Asn Val Gln Ile Asn Asp Le #u Thr Cys Gly Pro Gly        275           #       280           #       285His Gly Ile Ser Ile Gly Ser Leu Gly Asp As #p Asn Ser Lys Ala Tyr    290               #   295               #   300Val Ser Gly Ile Asp Val Asp Gly Ala Thr Le #u Ser Glu Thr Asp Asn305                 3 #10                 3 #15                 3 #20Gly Val Arg Ile Lys Thr Tyr Gln Gly Gly Se #r Gly Thr Ala Lys Asn                325   #               330   #               335Ile Lys Phe Gln Asn Ile Arg Met Asp Asn Va #l Lys Asn Pro Ile Ile            340       #           345       #           350Ile Asp Gln Asn Tyr Cys Asp Lys Asp Lys Cy #s Glu Gln Gln Glu Ser        355           #       360           #       365Ala Val Gln Val Asn Asn Val Val Tyr Gln As #n Ile Lys Gly Thr Ser    370               #   375               #   380Ala Thr Asp Val Ala Ile Met Phe Asn Cys Se #r Val Lys Tyr Pro Cys385                 3 #90                 3 #95                 4 #00Gln Gly Ile Val Leu Glu Asn Val Asn Ile Ly #s Gly Gly Lys Ala Ser                405   #               410   #               415Cys Glu Asn Val Asn Val Lys Asp Lys Gly Th #r Val Ser Pro Lys Cys            420       #           425       #           430 Pro<210> SEQ ID NO 3 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:oligo-      nucleotide PG1 <221> NAME/KEY: modified_base <222> LOCATION: (16)<223> OTHER INFORMATION: i <220> FEATURE:<223> OTHER INFORMATION: Location 22 and Location  #25 =  n = unknown.<400> SEQUENCE: 3 ccaggaattc aayacngayg gnrtnca          #                   #             27 <210> SEQ ID NO 4 <211> LENGTH: 25<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:oligo-      nucleotide PG2 <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (12) <223> OTHER INFORMATION: i <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: ()..)<223> OTHER INFORMATION: Location 21 and Location  #24 = n = a, c, g, t,      any, unknown, or other. <400> SEQUENCE: 4cgacggatcc angtyttdat nckna           #                  #               25 <210> SEQ ID NO 5 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:oligo-      nucleotide PG3 <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: ()..) <223> OTHER INFORMATION: any n = a, c, g,# t, any, unknown, or other <400> SEQUENCE: 5ggacgaattc acnggngayg aytgyat           #                  #             27 <210> SEQ ID NO 6 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:oligo-      nucleotide PG5 <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (13) <223> OTHER INFORMATION: i <220> FEATURE:<221> NAME/KEY: modified_base <222> LOCATION: (16)<223> OTHER INFORMATION: i <220> FEATURE: <221> NAME/KEY: modified_base<222> LOCATION: (19) <223> OTHER INFORMATION: i <400> SEQUENCE: 6cacaggatcc swngtnccny kdatrtt           #                  #             27 <210> SEQ ID NO 7 <211> LENGTH: 155 <212> TYPE: DNA<213> ORGANISM: Brassica napus <220> FEATURE: <221> NAME/KEY: unsure<222> LOCATION: (13)..(19)<223> OTHER INFORMATION: PCR fragment BPG32-26 fro #m first strand cDNA.      Strain cv. Topaz <223> OTHER INFORMATION: any n = a, c, g,# t, any, unknown, or other <400> SEQUENCE: 7tcgattcaaa ccggttgctc caatgtgtat gttcacaatg tgaattgtgg ac#caggacat     60ggcatcagca tagggagtct tggtaaagac agtaccaaag cttgtgtctc ca#atataaca    120 gtcagagatg tagttatgca caacacaatg actgg       #                   #      155 <210> SEQ ID NO 8 <211> LENGTH: 155<212> TYPE: DNA <213> ORGANISM: Brassica napus <220> FEATURE:<223> OTHER INFORMATION: PCR fragment KPG32-8 from # first strand cDNA.      Strain cv. Topaz. <400> SEQUENCE: 8tctattggag acgggacgag agaccttctt gtcgaaagag ttacatgcgg tc#cgggacat     60ggaatcagta ttggaagcct cggtttatac gtgaaggagg aagacgtcac tg#gcatcagg    120 gtcgtgaact gcaccctcat aaacactgac aatgg       #                   #      155 <210> SEQ ID NO 9 <211> LENGTH: 219<212> TYPE: DNA <213> ORGANISM: Brassica napus <220> FEATURE:<223> OTHER INFORMATION: PCR fragment LPG12-16 fro #m first strand cDNA.      Strain cv. Topaz. <400> SEQUENCE: 9tttgggaaga agtgacggag tcaagatcct taacacattc atctccaccg ga#gacgactg     60tatctccgtt ggagatggga tgaagaacct tcacgtggag aaagtcacct gc#ggtccagg    120acatggaatc agtgtcggaa gccttggaag gtacggaaac gaacaggatg tc#agcggcat    180 tagagtcata aactgcactc tccaacagac tgacaacgg      #                   #   219 <210> SEQ ID NO 10 <211> LENGTH: 155<212> TYPE: DNA <213> ORGANISM: Brassica napus <220> FEATURE:<223> OTHER INFORMATION: PCR fragment LPG32-24 fro #m first strand cDNA.      Strain cv. Topaz. <400> SEQUENCE: 10tccattggag gcggtactga aaatttactt gtcgagggcg tagaatgtgg ac#caggacac     60ggtctttcca tcggaagtct tggaaagtac cctaatgagc aaccagtgaa ag#gaatcacc    120 attcgtaaat gcatcatcaa gcataccgat aatgg       #                   #      155 <210> SEQ ID NO 11 <211> LENGTH: 155<212> TYPE: DNA <213> ORGANISM: Brassica napus <220> FEATURE:<223> OTHER INFORMATION: PCR fragment LPG32-25 fro #m first strand cDNA.      Strain cv. Topaz. <400> SEQUENCE: 11tctgttgggg acgggatgaa aaaccttctt gtcgaaagag tttcatgcgg tc#cgggacac     60ggaatcagta ttggaagcct cggattatac gggcacgagg aagacgtcac tg#gcgtcaag    120 gtcgtgaact gcaccctcag aaatactgac aatgg       #                   #      155 <210> SEQ ID NO 12 <211> LENGTH: 155<212> TYPE: DNA <213> ORGANISM: Brassica napus <220> FEATURE:<223> OTHER INFORMATION: PCR fragment LPG32-32 fro #m first strand cDNA.      Strain cv. Topaz. <400> SEQUENCE: 12tccgtgggag atgggatgaa gaatctcctc attgagaaag ttgtgtgcgg tc#caggacac     60ggaatcagtg ttggaagcct tggaaggtac ggatgggagc aagatgtcac tg#acattaac    120 gttaagaact gtaccctcga gggaaccgac aacgg       #                   #      155 <210> SEQ ID NO 13 <211> LENGTH: 100<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence:DNA sequence       of the T-DNA of pGSV5<223> OTHER INFORMATION: Location 1-25 = right #border (RB) sequence from       the T-DNA of pGSV5.<223> OTHER INFORMATION: Location 26-75 = multi #ple cloning site (MCS).<220> FEATURE: <223> OTHER INFORMATION: Location 76-100 = left# border (LB) sequence       from the T-DNA of pGSV5. <400> SEQUENCE: 13aattacaacg gtatatatcc tgccagtact cggccgtcga ccgcggtacc cg#gggaagct     60 tagatccatg gagccattta caattgaata tatcctgccg     #                   #   100 <210> SEQ ID NO 14 <211> LENGTH: 2352<212> TYPE: DNA <213> ORGANISM: Brassica napus <220> FEATURE:<223> OTHER INFORMATION: Location 2329-2331 = t #ranslation initiation      codon. <223> OTHER INFORMATION: Location 246-251 = Sph#I restriction enzyme       recognition site.<223> OTHER INFORMATION: Location 2219-2227 = t #ranscript_star = region      containing the putative location of  #transcription      start site. <223> OTHER INFORMATION: Strain cv. Bridger.<223> OTHER INFORMATION: Location 1836-1841 = H#indII restriction enxyme       recognition site.<223> OTHER INFORMATION: Location 2327-2332 = s#equence mutated to form a       NcoI restrication enzyme recognition #site  (AAATGG       changed to CCATGG). <400> SEQUENCE: 14acgagaatcg agagaaacaa aaactctcgt cgcaagcaca agtttggggt ag#gttgtatg     60gtgtaagaat gacgggccat agagaataat gtcttccact cttgccaaac gg#actgaaac    120catcagtaca taatccaagg tagacatttc ttctctcata cgcaaagtcg gg#atactttg    180attggaaatg cttccacgct tttgcatctg aaggatgtct gatctcacca tc#tgttgagt    240gctccgcatg ccatctcatt ggttgcgctg tgcgttcaga cagatacaac ct#ctgcaacc    300tttccgtcaa aggtaaatac cacatccttt tatatggcac tggaactctt cc#actcgtat    360ctttataacg aggctttcca caaaatttgc atgtaacccg ctgttcatcc gc#cctccaat    420aaatcatgca gttgtcgctg catacatcta ttacctgata cgccaagacc ag#ctacgagt    480ttctgaacct cgtagtatga accaggagct acattattct cgggtagaat ac#cttttaca    540aaatcagcaa tcgcatccac acagtcttca gccaaattat aatctttttc aa#tgcccatc    600aatcttgtag cagatgataa agctgaatga ccatctctgc aaccttcgta ca#atggttgc    660tttccagcat ccaacatatc ataaaatttc ctagcttgtg cattgggtaa at#cttcccct    720ctaaaatgat catttaccat ctgctcagta cctacaccat aatctacatc cg#ttctaatt    780ggttcttcta atctaaccgc tggctgaggt tcgctagtac taccatgttc at#aatcagtt    840tccccatgat gataccaaat tttgtaactt cgtgtaaacc cactcaaata ta#gatgagtc    900caaacatccc actctttaat aacttttcta tttttacaat tagagcaaga ac#atcttaac    960atacatgttt ttgcttccgg ttgtcggtga actaacccca tgaattcggt ta#tacctcgt   1020tggtattctt ccgtaagcaa tctcgtgttc ggatccaaat gaggtcgatc ga#tccaagaa   1080cgaaaataat ttgaagaaga catatttttt atgaatcaaa ttcgtgtgta aa#tagagtaa   1140gagggaggat gaagatatgg agtgaatgaa gaggaagagg agtgcttgta tt#tatagttt   1200aaatcctgcc gacagaccga ggaaattccg acggaattcc gacggaaaag gc#tagttcgt   1260cggaatttcc tcggaatttt gtaaaatccc ccagcggctc tccaacggct at#aatatttc   1320ctcggaattc atcggttttt tccgaggaac acatttttcc tcggaatttc ct#cggaatat   1380tccaacggat tgatatttcc tcggaattcc gtcggtatat tccaaggaaa cc#caattttg   1440tgtttcctca gaatttcctc agaaattcct cgggatattc cgaggatttc at#tttccgtc   1500ggaatgtcca tcagaatacc gctgttttct tgtagtgatt attatttttt tt#ttcagata   1560aaaaaaaaag aaatatcaac caatcgctga ctgtcacata ttgtgggggc cc#acaaatag   1620tgcaaggact cactaagaaa aagtttttat tttagaattt tagtaattga at#tcttaact   1680tttggtggag ttcactgatt atttaattat ttttttttaa gaactcaccc tt#aagaattg   1740ttgttgcgga tgttctaata tcagcatcac acaccaaaat aaaaagcacg aa#agagtaaa   1800agggacccaa cactactatc gaactttgaa agacggttga cgccgacgtt ta#tcactttt   1860gcttatatgt tttcaacttt ttatatctaa tgtagggata tatacatcac gt#aatgttag   1920ctcagtaatt gcacatgatg gaatgttact gtgaatggta tacgatgatg aa#tataaact   1980cttttctagt agaaaataac taactaatta aactctctat caatcaagaa ag#caataaaa   2040atcaataaaa agataaatta aaatggaggg gagaggagat aaaggttaga ag#ctagggtg   2100tgatgttttc gtatcaatct caatctctct ccatacctcc aacgccatta at#acttgaat   2160aaacatataa aatttctcca ttgaattgcc tataaataca catacatccc ac#ttcttcaa   2220tttcatatta caaaagcctc ccaaaaactg caaagagtct catattagtt ct#tactctca   2280agaatcaaac acactctttc taaaaagatt agcgtttcaa accccgaaat gg#cccgttgt   2340 tttggaagtc ta               #                  #                   #     2352

What is claimed is:
 1. An isolated DNA fragment comprising thenucleotide sequence of SEQ ID NO:1 from the nucleotide at position 10 tothe nucleotide at position
 1600. 2. An isolated DNA fragment comprisinga nucleotide sequence encoding an endopolygalacturonase comprising theamino acid sequence of SEQ ID NO: 2, wherein the sequence from the aminoacid at position 1 to the amino acid at position 24 of SEQ ID NO: 2 is asignal peptide.
 3. An isolated DNA fragment comprising a nucleotidesequence encoding an endopolygalacturonase comprising the amino acidsequence of SEQ ID NO:2 from the amino acid at position 24 to the aminoacid at position
 433. 4. An isolated DNA fragment comprising anucleotide sequence encoding an endopolygalacturonase comprising theamino acid sequence of SEQ ID NO:2 from the amino acid at position 1 tothe amino acid at position
 433. 5. A vector comprising the DNA fragmentaccording to any one of claim 1, 2, 3 or
 4. 6. A chimeric DNA constructcomprising the following operably linked elements: (a) a promoter whichis at least active in the pod dehiscence zone of a Brassica plant; and(b) a DNA fragment encoding an antisense RNA, wherein said DNA fragmentcomprises the complement of a nucleotide sequence encoding SEQ ID NO: 2.7. A chimeric DNA construct comprising the following operably linkedelements: (a) a promoter which is at least active in the pod dehiscencezone of a Brassica plant; and (b) a DNA fragment encoding an antisenseRNA, wherein said DNA fragment comprises the complement of thenucleotide sequence of SEQ ID NO:
 1. 8. A chimeric DNA constructcomprising the following operably linked elements: (a) a promoter whichis at least active in the pod dehiscence zone of a Brassica plant; and(b) a DNA fragment encoding a sense RNA, wherein said DNA fragmentcomprises a nucleotide sequence encoding SEQ ID NO:
 2. 9. A chimeric DNAconstruct comprising the following operably linked elements: (a) apromoter which is at least active in the pod dehiscence zone of aBrassica plant; and (b) a DNA fragment encoding a sense RNA, whereinsaid DNA fragment comprises the nucleotide sequence of SEQ ID NO:
 1. 10.An oil seed rape plant with delayed pod-dehiscence comprising thechimeric DNA construct according to any one of claim 6, 7, 8, or 9incorporated in its genome.
 11. An oil seed rape plant cell comprisingthe chimeric DNA construct according to any one of claim 6, 7, 8, or 9incorporated in its genome.
 12. An oil seed rape plant seed comprisingthe chimeric DNA construct according to any one of claim 6, 7, 8, or 9incorporated in its genome.
 13. A method for producing an oil seed rapeplant with delayed pod dehiscence, comprising: (a) transforming an oilseed rape plant cell with the chimeric DNA construct according to anyone of claim 6, 7, 8, or 9; and (b) regenerating a transformed oil seedrape plant from said transformed plant cell.
 14. The chimeric DNAconstruct according to any one of claim 6, 7, 8 or 9, wherein thepromoter is a constitutive promoter or a dehiscence zone specificpromoter.
 15. A Brassica plant with delayed pod-dehiscence comprisingthe chimeric DNA construct according to any one of claim 6, 7, 8, or 9incorporated in its genome.
 16. A Brassica plant cell comprising thechimeric DNA construct according to any one of claim 6, 7, 8, or 9incorporated in its genome.
 17. A Brassica seed comprising the chimericDNA construct according to any one of claim 6, 7, 8, or 9 incorporatedin its genome.
 18. A method for producing a Brassica plant with delayedpod dehiscence, comprising: (a) transforming a Brassica plant cell withthe chimeric DNA construct according to any one of claim 6, 7, 8, or 9;and (b) regenerating a transformed Brassica plant from said transformedplant cell.